Targeting of Notch3 Receptor Function for Cancer Therapy

ABSTRACT

The present invention involves the use of peptides from Notch3, and antibodies that recognize epitopes represented by those peptides, as anti-cancer agents. Methods of combination therapy using standard anti-cancer protocols in conjunction with Notch3 peptides and antibodies also are provided.

This application claims benefit of priority to U.S. Prov. Appln. Ser.No. 60/972,584, filed Sep. 14, 2007, the entire contents of which arehereby incorporated by reference.

This invention was made with government support under grant no. 2P50CA090949 awarded by the National Cancer Institute. The government hascertain rights in the invention.

BACKGROUND OF THE INVENTION

I. Field of the Invention

The present invention relates to the fields of oncology and molecularbiology. More particular the invention relates to the targeting of theNotch3 receptor.

II. Related Art

Lung cancer is the most common cause of cancer-related deaths in theUnited States. The cure rate for patients with lung cancer remainslow—15%—and has not changed significantly during the past 30 years(Jermal et al., 2005). A better understanding of the signaling pathwaysimportant in driving and maintaining the malignant state allows theidentification of new therapeutic targets and is thus imperative forcontinued progress in the treatment of these patients. Genes involved incell fate determination often contribute to tumorigenesis when they areaberrantly expressed. The family of Notch receptors is one such familywhere there are now strong data linking it to cancer pathogenesis.

All four members of the Notch receptor family are known to bedysregulated in the majority of human cancers. The inventors were thefirst to link dysregulation of the Notch3 pathway to human lung cancer(Dang et al., 2000). They demonstrated that Notch3 is highly expressedin 40% of all resected lung cancers and that, in the developing lung,constitutive activation of Notch3 results in inhibition of terminaldifferentiation. Furthermore, they showed that inhibiting this pathwayin human lung tumors results in the loss of the malignant phenotype invitro and tumor inhibition in xenograft models. This anti-tumor effectis enhanced in the presence of low serum and in combination with an EGFrtyrosine kinase inhibitor. Taken together, these data support animportant role for Notch3 and its interaction with the EGF and Raspathways in lung cancer. However, methods for therapeutic interventionin Notch3 related cancers has not yet been reported.

SUMMARY OF THE INVENTION

Thus, in accordance with the present invention, there is provided anisolated and purified peptide of no more than about 50 residues andcomprising the sequence of CFNTLGGHS (SEQ ID NO:3), CVCVNGWTGES (SEQ IDNO:4), CATAV (SEQ ID NO:5), CFHGAT (SEQ ID NO:6), CVSNP (SEQ ID NO:7) orCLNGGS (SEQ ID NO:8). More particular peptides include the sequencesCFNTLGGHSCVCVNGWTGESCSQNIDDCATAVCFHGAT (SEQ ID NO:9) orCTNLAGSFSCTCHGGYTGPSCDQDINDCDPNPCLNGGS (SEQ ID NO:10). The peptide maybe no more than about 25 residues, or no more than about 20 residues, orno more than about 15 residues. The peptide may consist of the sequenceof CFNTLGGHS (SEQ ID NO:3), CVCVNGWTGES (SEQ ID NO:4), CATAV (SEQ IDNO:5), CFHGAT (SEQ ID NO:6), CVSNP (SEQ ID NO:7), CLNGGS (SEQ ID NO:8),CFNTLGGHSCVCVNGWTGESCSQNIDDCATAVCFHGAT (SEQ ID NO:9) orCTNLAGSFSCTCHGGYTGPSCDQDINDCDPNPCLNGGS (SEQ ID NO:10). The peptide mayfurther be comprised in a pharmaceutically acceptable diluent, buffer orexcipient.

In another embodiment, there is provided a method of inhibiting Notch3receptor signaling comprising contacting a cell expressing Notch3 with apeptide of no more than about 50 residues and comprising the sequence ofCFNTLGGHS (SEQ ID NO:3), CVCVNGWTGES (SEQ ID NO:4), CATAV (SEQ ID NO:5),CFHGAT (SEQ ID NO:6), CVSNP (SEQ ID NO:7) or CLNGGS (SEQ ID NO:8). Thecell may be a cancer cell, such as a lung cancer cell and/or anadenocarcinoma. More particular peptides include the sequencesCFNTLGGHSCVCVNGWTGESCSQNIDDCATAVCFHGAT (SEQ ID NO:9) orCTNLAGSFSCTCHGGYTGPSCDQDINDCDPNPCLNGGS (SEQ ID NO:10). The peptide maybe no more than about 25 residues, or no more than about 20 residues, orno more than about 15 residues. The peptide may consist of the sequenceof CFNTLGGHS (SEQ ID NO:3), CVCVNGWTGES (SEQ ID NO:4), CATAV (SEQ IDNO:5), CFHGAT (SEQ ID NO:6), CVSNP (SEQ ID NO:7), CLNGGS (SEQ ID NO:8),CFNTLGGHSCVCVNGWTGESCSQNIDDCATAVCFHGAT (SEQ ID NO:9) orCTNLAGSFSCTCHGGYTGPSCDQDINDCDPNPCLNGGS (SEQ ID NO:10). The method mayfurther comprise contacting the cell with two or more peptidescomprising sequences of at least two of CFNTLGGHS (SEQ ID NO:3),CVCVNGWTGES (SEQ ID NO:4), CATAV (SEQ ID NO:5), CFHGAT (SEQ ID NO:6),CVSNP (SEQ ID NO:7), CLNGGS (SEQ ID NO:8),CFNTLGGHSCVCVNGWTGESCSQNIDDCATAVCFHGAT (SEQ ID NO:9) orCTNLAGSFSCTCHGGYTGPSCDQDINDCDPNPCLNGGS (SEQ ID NO:10). The method mayalso further comprises contacting the cancer cell with a second agentthat inhibits cancer cell growth, differentiation, metastasis or drugresistance.

In yet another embodiment, there is provided a method of treating asubject having a Notch3-expressing cancer comprising administering tosaid subject a peptide of no more than about 50 residues and comprisingthe sequence of CFNTLGGHS (SEQ ID NO:3), CVCVNGWTGES (SEQ ID NO:4),CATAV (SEQ ID NO:5), CFHGAT (SEQ ID NO:6), CVSNP (SEQ ID NO:7) or CLNGGS(SEQ ID NO:8). The subject may a human. The cancer cell may be a lungcancer cell and/or an adenocarcinoma. More particular peptides includethe sequences CFNTLGGHSCVCVNGWTGESCSQNIDDCATAVCFHGAT (SEQ ID NO:9) orCTNLAGSFSCTCHGGYTGPSCDQDINDCDPNPCLNGGS (SEQ ID NO:10). The peptide maybe no more than about 25 residues, or no more than about 20 residues, orno more than about 15 residues. The peptide may consist of the sequenceof CFNTLGGHS (SEQ ID NO:3), CVCVNGWTGES (SEQ ID NO:4), CATAV (SEQ IDNO:5), CFHGAT (SEQ ID NO:6), CVSNP (SEQ ID NO:7), CLNGGS (SEQ ID NO:8),CFNTLGGHSCVCVNGWTGESCSQNIDDCATAVCFHGAT (SEQ ID NO:9) orCTNLAGSFSCTCHGGYTGPSCDQDINDCDPNPCLNGGS (SEQ ID NO:10). The method mayfurther comprise contacting the cell with two or more peptidescomprising sequences of at least two of CFNTLGGHS (SEQ ID NO:3),CVCVNGWTGES (SEQ ID NO:4), CATAV (SEQ ID NO:5), CFHGAT (SEQ ID NO:6),CVSNP (SEQ ID NO:7), CLNGGS (SEQ ID NO:8),CFNTLGGHSCVCVNGWTGESCSQNIDDCATAVCFHGAT (SEQ ID NO:9) orCTNLAGSFSCTCHGGYTGPSCDQDINDCDPNPCLNGGS (SEQ ID NO:10). The method mayalso further comprises contacting the cancer cell with a second agentthat inhibits cancer cell growth, differentiation, metastasis or drugresistance.

Also provided are an isolated and purified antibody that binds to anepitope comprising the sequence of CFNTLGGHS (SEQ ID NO:3), CVCVNGWTGES(SEQ ID NO:4), CATAV (SEQ ID NO:5), CFHGAT (SEQ ID NO:6), CVSNP (SEQ IDNO:7) or CLNGGS (SEQ ID NO:8), as well as methods of using suchantibodies to inhibit Notch3 receptor signaling in a cell expressingNotch3.

In still another embodiment, there is provided a method of treating asubject having a Notch3-expressing cancer comprising administering tosaid subject an antibody that binds to an epitope comprising thesequence of CFNTLGGHS (SEQ ID NO:3), CVCVNGWTGES (SEQ ID NO:4), CATAV(SEQ ID NO:5), CFHGAT (SEQ ID NO:6), CVSNP (SEQ ID NO:7) or CLNGGS (SEQID NO:8).

Yet another embodiment comprises a pharmaceutical formulation comprisingtwo or more of CFNTLGGHS (SEQ ID NO:3), CVCVNGWTGES (SEQ ID NO:4), CATAV(SEQ ID NO:5), CFHGAT (SEQ ID NO:6), CVSNP (SEQ ID NO:7), CLNGGS (SEQ IDNO:8), CFNTLGGHSCVCVNGWTGESCSQNIDDCATAVCFHGAT (SEQ ID NO:9) orCTNLAGSFSCTCHGGYTGPSCDQDINDCDPNPCLNGGS (SEQ ID NO:10) including three,four, five, six, seven or all of CFNTLGGHS (SEQ ID NO:3), CVCVNGWTGES(SEQ ID NO:4), CATAV (SEQ ID NO:5), CFHGAT (SEQ ID NO:6), CVSNP (SEQ IDNO:7), CLNGGS (SEQ ID NO:8), CFNTLGGHSCVCVNGWTGESCSQNIDDCATAVCFHGAT (SEQID NO:9) or CTNLAGSFSCTCHGGYTGPSCDQDINDCDPNPCLNGGS (SEQ ID NO:10).

It is contemplated that any method or composition described herein canbe implemented with respect to any other method or composition describedherein.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.”

These, and other, embodiments of the invention will be betterappreciated and understood when considered in conjunction with thefollowing description and the accompanying drawings. It should beunderstood, however, that the following description, while indicatingvarious embodiments of the invention and numerous specific detailsthereof, is given by way of illustration and not of limitation. Manysubstitutions, modifications, additions and/or rearrangements may bemade within the scope of the invention without departing from the spiritthereof, and the invention includes all such substitutions,modifications, additions and/or rearrangements.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein:

FIGS. 1A-B. Diagram demonstrates the major steps of the canonical Notchpathway. (FIG. 1A) Binding of a DSL ligand triggers proteolysis (S2 andS3) of the Notch receptor and releases the C-terminal NICD fragment.(FIG. 1B) NICD translocates to the nucleus, recruits coactivators, andbinds to CSL factors to promote target gene transcription. In theabsence of NICD, the CSL factor associates with the corepressor complexand inhibits target gene transcription.

FIGS. 2A-B. Proposed mechanisms of antagonistic and cooperativeinteractions between Ras and Notch pathways. (FIG. 2A) Notch antagonizesRas signaling by preventing expression of active EGF (1). Notchactivation can also directly inhibit Ras activity (2) and induceexpression MAPK inhibitors (3). (FIG. 2B) Ras activation in turn caninduce expression of Notch inhibitors (4). Paradoxically, Ras activationhas also been shown to induce DSL ligand expression (5). Theseobservation further support the hypothesis that Notch activation ishighly context dependent. However, in a majority of cancers, Notch andras appear to be cooperative. Adapted from Sundaram (2005).

FIG. 3. Immunohistochemistry with an antibody to extracellular domain ofNotch3. Cytoplasmic and membranous staining is observed in squamous cellcarcinoma (A) and adenocarcinoma (C) of the lung as compared toaneuroendocrine tumor (B) and normal lung (D). In panel B, slightstaining is seen in blood vessels (arrow) within the tumor, consistentwith other studies demonstrating normal staining of Notch3 in bloodvessels

FIGS. 4A-D. Notch3 alters lung morphology of SP-C-N3IC transgenic miceat E18.5. Five μm-thick lung sections of wild-type littermate controls(FIGS. 4A, 4C) show that epithelial layer of terminal airways are thinand comprised mostly of type I pneumocytes. The terminal lung epitheliumof transgenic embryos (FIGS. 4B, 4D) demonstrates severe metaplasia,composed mostly of undifferentiated cuboidal cells (arrowheads). No typeI pneumocyte was found. The mesenchyme is abnormally abundant comparedto that seen in the wild-type littermate. Bars=50 μm; br., bronchiole;m, mesenchyme; v, vessel.

FIGS. 5A-B. Notch inhibition inhibits the tumor phenotype. (FIG. 5A)Inhibition of the Notch3 signaling pathway markedly reduces the size ofthe colonies formed in soft agar (panel B), compared with vectorcontrols (panel A) in HCC2429 and H460. (FIG. 5B) In serum-starvedconditions, the growth of the DN transfectant is severely inhibited incomparison with that of VC. However, with the addition of exogenousgrowth factors, the growth rate is equal to that of VC.

FIG. 6. Knocking out Notch3 with siRNA reduces focus formation. ThepSuper vectors expressing Notch3 siRNA and a mouse Notch3 sequence weretransfected into HCC2429, a human lung cancer cell line. The cells wereselected with puramycin, then stained with crystal violet after 4 weeks.

FIGS. 7A-D. A γ-secretase inhibitor inhibits tumor cells and Notch3processing. (FIG. 7A) GSI inhibits HCC2429 cells in a serum-dependentmanner. HCC2429 cells are sensitive to GSI, and the sensitivityincreases in low serum, similarly to that observed with clonesexpressing the DN construct. Tumor cells treated with DMSO alone had nochange in cell viability. (FIG. 7B) Inhibition of S3 proteolyticprocessing results in the decrease of Notch3 intracellular domain(N3ICD) and accumulation of S2 product (N3ΔE) after 3 hours. (FIG. 7C)In this experiment, HCC2429 was stably transfected with plasmid vectorexpressing Notch3 siRNA. Control C is the parental HCC2429, whereassiRNA-C clones 5, 6, 8 expressed high level of HCC2429 and siRNA-N3clones 12, 15, 17 and 20 expressed significantly lower level of Notch3(FIG. 7D). Loss of Notch3 results in no loss in cell survival whentreated with MRK003.

FIGS. 8A-D. γ-secretase inhibitor MRK003 demonstrates anti-tumoractivity in vivo. (FIG. 8A) Xenografts injected with HCC2429 weretreated with MRK003 once the tumors became palpable. After 2 weeks oftreatment, the inventors observed about a 50% reduction in tumor size.(FIG. 8B) Loss of activated Notch3 (N3ICD) can be seen in tumor treatedwith MRK003. Histological examination of resected tumors from xenograftsat the end of treatment. Marked necrosis can be seen in the MRK003treated animal (FIG. 8D) as compare to control (FIG. 8C).

FIGS. 9A-C. Inhibition of Notch3 increases apoptosis. (FIG. 9A) After 72hours of exogenous growth factor deprivation, cell lines transfectedwith DN show a higher percentage of apoptosis as measured by Apo-BrdUanalysis. (FIG. 9B) Expression levels of phospho-Akt protein decrease inthe Notch3-overexpressing cell line HCC2429 when it is stablytransfected with the DN construct, particularly with serum starvation.(FIG. 9C) Transfection with Notch3 SiRNA resulted in loss of Bcl-xLexpression and induction of apoptotic product PARP.

FIGS. 10A-C. Notch3 crosstalks with the MAPK pathway. (FIG. 10A)Inhibition of Notch3 signaling in HCC2429 downregulates phospho-p44/42(ERK?) under serum-starved conditions and after induction with 10% FCS.(FIG. 10B) When the immortalized lung epithelial cell line BEAS-2B wastransfected with the DA construct, the inventors observed higher levelsof phospho-p44/p42 under serum-starved conditions as well as after seruminduction. (FIG. 10C) One mechanism of MAPK modulation includestranscriptional regulation of MKP1 in HCC2429. The DN clones demonstratesignificantly higher transcriptional level of MKP1 under serum-starvedconditions and at 30 minutes and 1 hour after serum induction, whencompared with VC (*).

FIGS. 11A-B. Notch3 modulates the EGF pathway and increases sensitivityto an EGFr inhibitor. (FIG. 11A) In HCC2429, inhibition of the Notch3pathway increases sensitivity to AG1478 nearly 40-fold. In H460, a cellline that is markedly resistant to AG1478 (IC₅₀=23.8 μM) when comparedto HCC2429 (IC₅₀=8.3 μM), inhibition of Notch3 also increasessensitivity to the inhibitor. (FIG. 11B) A similar observation is madein H460 when the inventors combine AG1478 with L-685,458, a γ-secretaseinhibitor, further supporting the hypothesis that EGF cooperates withthe Notch pathway in oncogenesis.

FIGS. 12A-B. In HCC2429 lung cancer cell line, Notch inhibitor MRK003enhances the effect of EGFR inhibitor AG1478 on colony formation in softagar. (FIG. 12A) Photographs showing that MRK003 not only decreasescolony formation, but also enhances the effect of AG1478 on growth.(FIG. 12B) Graph depicts the quantitative decrease in colony formation.

FIG. 13. Notch3 peptides induce apoptosis. Representative experimentshowing that the peptides induce apoptosis by Annexin V staining throughscreening using an FMAT system. Each of 155 different peptides wereassayed in quadruplicate, and only those peptides that produced asignificant increase of fluorescence signal in all 4 wells wereconsidered potentially positive or capable of inducing apoptosis. Thebar graphs here reflect fluorescence counts. Sequences N102: CATAV,N103: CFHGAT, N105: CVSNP, N132: CLNGGS.

FIG. 14A-B. Notch3 peptides induce apoptosis and inhibitNotch3-regulated gene Hey1. (FIG. 14A) HCC2429 was treated with Notch3peptides N16, N17, N102, N103, N132. Induction of apoptosis by peptidesis observed as compared to control. MRK003-treated cell is used aspositive control. After treatment, cells were labeled with annexin V anddetected using flow cytometry. (FIG. 14B) Treatment with peptides alsoreduced transcription of Notch3-dependent gene Hey1 as determined byreal-time RT-PCR. Of note, N17 peptide both demonstrates highestapoptotic activity and best reduction in Hey1 transcription.MRK003-treated cells were used as positive control. Sequences N16:CFNTLGGHS, N17: CVCVNGWTGES, N102: CATAV, N103: CFHGAT, N132: CLNGGS.

FIG. 15. Notch3 Peptides interrupt signaling through binding to ligandJagged1. HEK cells were transfected with Jagged1-HA and treated withNotch3 peptides. The peptides were then immunoprecipitated from celllysate with streptavidin beads and immunoblotted with anti-HA antibody.No: no input; C: control peptide. Sequences N16: CFNTLGGHS, N17:CVCVNGWTGES, N102: CATAV, N103: CFHGAT, N132: CLNGGS.

FIG. 16. Sera from mice immunized with Notch3 recombindant proteininhibit Notch3 activation. Immunoblot demonstrates sera from mice #2, 3,4, 5, 6 can reduced cleavage of Notch3 ICD (Tx) in Notch3 expressingcell line HCC2429 as compared control (C). Recombinant proteinrepresenting EGF-like repeats 21-22 and encompassing sequenceCTNLAGSFSCTCHGGYTGPSCDQDINDCDPNPCLNGGS was used to immunize AJ andBALB/c mice. HCC2429 was plated in a 6-well plates and treated 1 μl/mlsera for 24 hr before harvesting.

FIGS. 17A-B. Recombinant Fc-fusion Notch3 proteins inhibit Notch3activation and induces apoptosis in vitro. (FIG. 17A) Fc-fusion proteincomprised for N16-17 and N132 sequences inhibits Notch3 activation.HCC2429 was treated with purified Fc-fusion protein 10 μg/ml for 24 hrs.(FIG. 17B) Purified recombinant N16-17-Fc protein induces apoptosis ascompared to control and Fc control after 40 hrs treatment. Apoptosis wasdetermined by percentage of annexin V positive cells. Sequences N16:CFNTLGGHS, N17: CVCVNGWTGES, N132: CLNGGS.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS I. The PresentInvention

Many genes that are important in tumor initiation, progression, orsurvival play crucial roles in normal development. The Notch receptorsare members of an evolutionarily conserved family that is essential forthe control of cell fate determination during the development of manymulticellular organisms. The core components of the Notch pathway arelisted in Table 1. Their functions were first discovered in Drosophilamelanogaster almost 80 years ago, when a heterozygous deletion was foundto result in “notches” at the wing margins. Mutations in Notch genesalter cell fate determination, causing cells destined to becomeepidermis to instead give rise to neural tissue (reviewed inArtavanis-Tsakonas, 1999). Notch signaling is classically divided intotwo fundamental types: inductive and lateral signaling. Induction occursbetween two nonequivalent cells, where one cell expresses the receptorand the other expresses the ligand, and through their interaction onecell adopts a different fate. In contrast, lateral signaling occursbetween equivalent cells, and through competitive inhibition, adjacentcells are forced to follow a different fate. The role of Notch pathwaysignaling in mammals has been studied extensively in lymphogenesis,ondotogenesis, neurogenesis, hair and sensory development (Beatus andLeandahl, 1998; Mitsiadis et al., 1998; Lanford et al., 1999; Robey etal., 1996).

TABLE 1 Core components of the Notch pathway in worms, flies, andmammals Component C. elegans Drosophila Mammals DSL Ligands LAG-2,APX-1, DSL-1 Delta, Serrate Delta-like-1, -3 and -4 Jagged1, Jagged 2Notch Receptors LIN-12, Glp-1 Notch Notch 1-4 CSL Factors LAG-1Suppressor of Hairless CBF-1 Corepressor Hairless, Groucho, SMRT, NcoR,CIR dCTBP, SMRTER Coactivators Mastermind Mastermind Target GenesEnhancer of Split, Hey HES1-7, Hey1-2 *adapted from Sundaram (2005).

II. Notch3

Notch Plays A Key Role In Vascular Development and Homeostasis. In adultmammals, the expression of Notch receptors is restricted to the vascularsystems. Mice with targeted mutations of the Notch pathway components,such as Jagged1, Delta-like-1 and the Notch1 receptor, die duringembryogenesis from defects in vascular morphogenesis (Krebs et al.,2000; Xue et al., 1999; Hrabe de Angelis et al., 1997). While Notch3−/−mice are fertile and viable, these adult mice exhibit structural defectsin the distal arteries and arterial myogenic response, reflecting thelack of proper development in vascular smooth muscle cells (Domenga etal., 2004). These observations indicate that the Notch signaling pathwayis important in both vascular development and homeostasis. Notsurprisingly, many of the processes involved in embryonic vasculardevelopment are mirrored in tumor angiogenesis. For example, inductionof Notch ligand Jagged1 promotes capillary-like sprout formation intumor cells (Zeng et al., 2005). Finally, inhibition of Notch activationby γ-secretase inhibitors also inhibits angiogenesis and tumorproliferation (Paris et al., 2005; Williams et al., 2005). Theseobservations support a role of Notch pathway in normal and tumorangiogenesis.

Activation of Notch Signaling requires Proteolytic Cleavage of theReceptor. While Drosophila possesses a single Notch gene, there are fourmembers of the Notch family in mammals: Notch1 (TAN1), Notch2, Notch3and Notch4/Int-4. The core components of the Notch pathway are listed inTable 1. Notch is expressed on cell surfaces as a single-pass,heterodimeric receptor. The ligands are also transmembrane proteins ofthe DSL (Delta/Serrate/LAG-2) family that can be expressed not only onadjacent cells but also on the very same cell expressing the Notchreceptors. Receptor-ligand interaction triggers proteolysis at theextracellular S2 site near the transmembrane domain and at the S3 site(FIG. 1). A TNF-α converting enzyme (TACE) and a presenillin-1-dependentγ-secretase are believed to be responsible for the proteolyticprocessing at sites S2 and S3, respectively. The final cleavage releasesthe C-terminal, intracellular domain (NICD), which then translocates tothe nucleus, recruits coactivators such as mastermind and p300, andbinds to CSL (CBF/Suppressor of Hairless/LAG-1) factors. In the absenceof Notch signaling, CSL proteins in association with corepressorsrepress target gene transcription. Thus, Notch signaling causes a switchfrom transcriptional repression to transcriptional activation of CSLtarget genes (a review of Notch processing in Mumm and Kopan (2000).

The Notch Signaling Pathway Is Oncogenic. Many key pathways indevelopment play important roles in tumorigenesis when altered. Notch1was first identified in association with a t(7:9) translocation found ina subset of human T-cell acute lymphoblastoid leukemias (T-ALLs)(Ellisen et al., 1991). While less than 1% of human T-ALLs exhibit thet(7:9), Notch activating mutations have been observed in 50% of humanT-ALLs (Weng et al., 2004; Ma et al., 1999). The expression of theconstitutively activated intracellular domain (NICD) of Notch1 in bonemarrow cells confers an oncogenic phenotype (Pear et al., 1996). Similarobservations have been made linking Notch family members with cancerpathogenesis as well (Jhappan et al., 1992; Rohn et al., 1996).Constitutive activation of Notch3 in transgenic mice results in T-celllymphoblastic leukemia, and in human T-ALL, loss of Notch3 expressioncorrelates with clinical remission Bellavia et al., 2002; Bellavia etal., 2000). Moreover, activated Notch3 confers resistance to apoptosisand loss of contact inhibition in smooth muscle cells (Wang et al.,2002; Sweeney et al., 2004; Campos et al., 2002). Notch3 has been foundto be highly expressed in other tumors, including lung, pancreatic andovarian carcinoma, using gene expression microarray (Dang et al., 2000;Miyamoto et al., 2003; Lu et al., 2004). Similar studies demonstratecorrelations between aberrant Notch ligand/receptor expression and tumordevelopment in various systems (Miyamoto et al., 2003; Santagata et al.,2004; Purow et al., 2005; Callahan and Egan, 2004). The inventorspublished data demonstrating that inhibition of Notch3 activation usinga dominant-negative receptor reduces tumor phenotype (Haruki et al.,2005). Taken together, these observations suggest that the Notch pathwayis functionally significant in solid tumors and can serve as a targetfor therapeutic intervention.

Notch Crosstalks with the Ras Pathway. In both mammals and invertebratessuch as Drosophila and C. elegans, Notch receptors signal primarily bythe binding to members of the CSL family of transcription factors andrelated transcription co-activators. However, Notch is known to interactwith other pathways including the Wingless/B-catenin and NF-κB pathways(Johnston and Edgar, 1998; Oswald et al., 1998). One pathway that playsprominently in both development and neoplastic transformation is theEGF/ras/MAPK pathway. Notch has been shown in developing organisms toantagonize EGF signaling in cell fate determination through modulationof the MAPK pathway (Faux et al., 2001; Ahmad and Dooley, 1998; Bersetet al., 2001). In other cases, however, the Ras and Notch pathwayscooperate in promoting certain cell fates (Yoo et al., 2004). As inflies and worms, specific outcomes of EGF and Notch pathways in mammalsare context dependent. In mammals, Wang et al. demonstrated that Notch3induces phosphorylation of ERK1/ERK2 (p44/p42) in vascular smooth musclecells (Wang et al., 2002). Current evidence indicates that malignanttransformation by Notch requires activated Ras (Haruki et al., 2005;Fitzgerald et al., 2000). On the other hand, activated Notch1 was foundto inhibit Fgf-dependent malignant transformation of NIH3T3 cells (Smallet al., 2003). While further work is required to fully understand themechanism of interactions between Ras and Notch pathways, thepreliminary data demonstrate a cooperative relationship between Notch3signaling and the ras pathway. This observation suggests thatcombinatorial therapeutic approach will have better efficacy in thetreatment of patients with lung cancers. FIG. 2 summarizes knownpotential Notch-ras interactions in both the development and cancercontext.

γ-Secretase Inhibitors Demonstrate Antitumor Effects. Proteolyticprocessing of Notch receptors following ligand binding is necessary fortheir activation. The final proteolytic cleavage by the γ-secretaseprotein complex releases the Notch intracellular domain required fortarget gene transcription. Thus, pharmacologic intervention thatinhibits the activity of any of the proteases can potentially inhibittumor growth in Notch-dependent cancer. Interestingly, at the same timethat presenilins were shown to be essential for Notch signaling, theywere discovered as susceptibility loci for Alzheimer's disease (Levitanand Greenwald, 1995). The pathogenesis of Alzheimer's disease isbelieved to be the accumulation of amyloid β-peptides (Aβ) and formationof amyloid plaques. These peptides are derived from the proteolyticprocessing of the β-amyloid precursor protein (APP) through anintermediate fragment (C99) by γ-secretases (Dovey et al., 2001). Giventhe great need for better treatment of patients with Alzheimer'sdisease, inhibitors targeting γ-secretase are being aggressively pursuedby many pharmaceutical companies. Predictably, many of these compoundswere found to inhibit Notch processing as well. Furthermore, γ-secretaseinhibitors block Notch activation and induce apoptosis in multiplecancer cell lines (Qin et al., 2004; Curry et al., 2005; Alves da Costa,2004). In vivo, these compounds inhibit angiogenesis and tumor growth(Paris et al., 2005). These inhibitors are known to have non-Notchtargets, such as erb-4 and CD44, but our data suggest that Notchinhibition may be a component of the observed antitumor effects(Pelletier et al., 2006; Linggi et al., 2006). However, from a practicalstandpoint, since erb-4 and CD44 are known to be oncogenic, theseinhibitors may actually have increased efficacy by virtue of theirmultiple targets. In fact, a γ-secretase inhibitor by Merck & Co., Inc,is currently in Phase I trials for patients with metastatic or locallyadvanced breast cancer and for patients with T-cell acute leukemias.

A. Features of the Polypeptide

Notch3 is a 2321 amino acid protein (243659 Da Q9UM47; SEQ ID NO:2) thatexists as a heterodimer of a C-terminal fragment (TM) and an N-terminalfragment (EC) which are probably linked by disulfide bonds. It has beenshown to iteract with MAML1, MAML2 and MAML3 which act astranscriptional coactivators for NOTCH3. It is localized in the cellmembrane and is a single-pass type I membrane protein. Followingproteolytical processing, the notch intracellular domain (NICD) causestranslocation to the nucleus. Its only known post-translationalmodification is glycosylation.

Notch3 functions as a receptor for membrane-bound ligands Jagged1,Jagged2 and Delta1 to regulate cell-fate determination. Upon ligandactivation through the released NICD, it forms a transcriptionalactivator complex with CBF-1 and activates genes of the enhancer ofsplit locus. As discussed above, it has effects the implementation ofdifferentiation, proliferation and apoptotic programs. It is located at19p13.2-p13.1.

B. Peptides

Notch3 peptides will comprise molecules of 5 to no more than about 50residues in length. A particular length may be less than 39 residues,less than 35 residues, less than 30 residues, less than 25 residues,less than 20 residues, less than 15 residues, or less than 13, including5, 6, 7, 8, 9, 10, 11 or 12 residues, and ranges of 5-11 residues, 5-15residues, 5-20 residues, 5-25 residues, 5-30 residues, 5-35 residues,5-38 residues, or 5-40 residues. The peptides may be generatedsynthetically or by recombinant techniques, and are purified accordingto known methods, such as precipitation (e.g., ammonium sulfate), HPLC,ion exchange chromatography, affinity chromatography (includingimmunoaffinity chromatography) or various size separations(sedimentation, gel electrophoresis, gel filtration), as described infurther detail below.

The peptides may be labeled using various molecules, such asfluorescent, chromogenic or colorimetric agents. The peptides may alsobe linked to other molecules, including other anti-cancer agents. Thelinks may be direct or through distinct linker molecules. The linkermolecules in turn may be subject, in vivo, to cleavage, therebyreleasing the agent from the peptide. Peptides may also be renderedmultimeric by linking to larger, and possibly inert, carrier molecules.

C. Variants

Amino acid sequence variants of the polypeptide can be substitutional,insertional or deletion variants. Deletion variants lack one or moreresidues of the native protein which are not essential for function orimmunogenic activity, and are exemplified by the variants lacking atransmembrane sequence described above. Another common type of deletionvariant is one lacking secretory signal sequences or signal sequencesdirecting a protein to bind to a particular part of a cell. Insertionalmutants typically involve the addition of material at a non-terminalpoint in the polypeptide. This may include the insertion of animmunoreactive epitope or simply a single residue. Terminal additions,called fusion proteins, are discussed below.

Substitutional variants typically contain the exchange of one amino acidfor another at one or more sites within the protein, and may be designedto modulate one or more properties of the polypeptide, such as stabilityagainst proteolytic cleavage, without the loss of other functions orproperties. Substitutions of this kind preferably are conservative, thatis, one amino acid is replaced with one of similar shape and charge.Conservative substitutions are well known in the art and include, forexample, the changes of: alanine to serine; arginine to lysine;asparagine to glutamine or histidine; aspartate to glutamate; cysteineto serine; glutamine to asparagine; glutamate to aspartate; glycine toproline; histidine to asparagine or glutamine; isoleucine to leucine orvaline; leucine to valine or isoleucine; lysine to arginine; methionineto leucine or isoleucine; phenylalanine to tyrosine, leucine ormethionine; serine to threonine; threonine to serine; tryptophan totyrosine; tyrosine to tryptophan or phenylalanine; and valine toisoleucine or leucine.

The following is a discussion based upon changing of the amino acids ofa protein to create an equivalent or improved molecule. For example,certain amino acids may be substituted for other amino acids in aprotein structure without appreciable loss of interactive bindingcapacity with structures such as, for example, antigen-binding regionsof antibodies or binding sites on substrate molecules. Since it is theinteractive capacity and nature of a protein that defines that protein'sbiological functional activity, certain amino acid substitutions can bemade in a protein sequence, and its underlying DNA coding sequence, andnevertheless obtain a protein with like properties. It is thuscontemplated by the inventors that various changes may be made in theDNA sequences of genes without appreciable loss of their biologicalutility or activity, as discussed below. Table 2 shows the codons thatencode particular amino acids.

TABLE 2 Amino Acids Codons Alanine Ala A GCA GCC GCG GCU Cysteine Cys CUGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu E GAA GAGPhenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGU Histidine HisH CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAA AAG Leucine LeuL UUA UUG CUA CUC CUG CUU Methionine Met M AUG Asparagine Asn N AAC AAUProline Pro P CCA CCC CCG CCU Glutamine Gln Q CAA CAG Arginine Arg RAGA AGG CGA CGC CGG CGU Serine Ser S AGC AGU UCA UCC UCG UCU ThreonineThr T ACA ACC ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGGTyrosine Tyr Y UAC UAU

In making substitutional variants, the hydropathic index of amino acidsmay be considered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte & Doolittle, 1982). It is accepted that therelative hydropathic character of the amino acid contributes to thesecondary structure of the resultant protein, which in turn defines theinteraction of the protein with other molecules, for example, enzymes,substrates, receptors, DNA, antibodies, antigens, and the like.

Each amino acid has been assigned a hydropathic index on the basis oftheir hydrophobicity and charge characteristics (Kyte & Doolittle,1982), these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8);phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9);alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8);tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2);glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5);lysine (−3.9); and arginine (−4.5).

It is known in the art that certain amino acids may be substituted byother amino acids having a similar hydropathic index or score and stillresult in a protein with similar biological activity, i.e., still obtaina biological functionally equivalent protein. In making such changes,the substitution of amino acids whose hydropathic indices are within ±2is preferred, those which are within ±1 are particularly preferred, andthose within ±0.5 are even more particularly preferred.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. U.S. Pat.No. 4,554,101, incorporated herein by reference, states that thegreatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with a biologicalproperty of the protein. As detailed in U.S. Pat. No. 4,554,101, thefollowing hydrophilicity values have been assigned to amino acidresidues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate(+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine(0); threonine (−0.4); proline (−0.5±1); alanine (−0.5); histidine*−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine(−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5);tryptophan (−3.4).

It is understood that an amino acid can be substituted for anotherhaving a similar hydrophilicity value and still obtain a biologicallyequivalent and immunologically equivalent protein. In such changes, thesubstitution of amino acids whose hydrophilicity values are within ±2 ispreferred, those that are within ±1 are particularly preferred, andthose within ±0.5 are even more particularly preferred.

As outlined above, amino acid substitutions are generally based on therelative similarity of the amino acid side-chain substituents, forexample, their hydrophobicity, hydrophilicity, charge, size, and thelike. Exemplary substitutions that take various of the foregoingcharacteristics into consideration are well known to those of skill inthe art and include: arginine and lysine; glutamate and aspartate;serine and threonine; glutamine and asparagine; and valine, leucine andisoleucine.

Another embodiment for the preparation of polypeptides according to theinvention is the use of peptide mimetics. Mimetics arepeptide-containing molecules that mimic elements of protein secondarystructure. See, for example, Johnson et al., (1993). The underlyingrationale behind the use of peptide mimetics is that the peptidebackbone of proteins exists chiefly to orient amino acid side chains insuch a way as to facilitate molecular interactions, such as those ofantibody and antigen. A peptide mimetic is expected to permit molecularinteractions similar to the natural molecule. These principles may beused, in conjunction with the principles outline above, to engineersecond generation molecules having many of the natural properties ofNotch3, but with altered and even improved characteristics.

D. Fusions

A specialized kind of insertional variant is the fusion protein. Thismolecule generally has all or a substantial portion (e.g., anintracellular, transmembrane or extracellular domain) of the nativemolecule, linked at the N- or C-terminus, to all or a portion of asecond polypeptide. For example, fusions typically employ leadersequences from other species to permit the recombinant expression of aprotein in a heterologous host. Another useful fusion includes theaddition of a immunologically active domain, such as an antibodyepitope, to facilitate purification of the fusion protein. Inclusion ofa cleavage site at or near the fusion junction will facilitate removalof the extraneous polypeptide after purification. Other useful fusionsinclude linking of functional domains, such as active sites fromenzymes, glycosylation domains, cellular targeting signals ortransmembrane regions.

E. Purification of Proteins/Peptides

It will be desirable to purify Notch3 or fragments thereof. Proteinpurification techniques are well known to those of skill in the art.These techniques involve, at one level, the crude fractionation of thecellular milieu to polypeptide and non-polypeptide fractions. Havingseparated the polypeptide from other proteins, the polypeptide ofinterest may be further purified using chromatographic andelectrophoretic techniques to achieve partial or complete purification(or purification to homogeneity). Analytical methods particularly suitedto the preparation of a pure peptide are ion-exchange chromatography,exclusion chromatography; polyacrylamide gel electrophoresis;isoelectric focusing. A particularly efficient method of purifyingpeptides is fast protein liquid chromatography or even HPLC.

Certain aspects of the present invention concern the purification, andin particular embodiments, the substantial purification, of an encodedprotein or peptide. The term “purified protein or peptide” as usedherein, is intended to refer to a composition, isolatable from othercomponents, wherein the protein or peptide is purified to any degreerelative to its naturally-obtainable state. A purified protein orpeptide therefore also refers to a protein or peptide, free from theenvironment in which it may naturally occur.

Generally, “purified” will refer to a protein or peptide compositionthat has been subjected to fractionation to remove various othercomponents, and which composition substantially retains its expressedbiological activity. Where the term “substantially purified” is used,this designation will refer to a composition in which the protein orpeptide forms the major component of the composition, such asconstituting about 50%, about 60%, about 70%, about 80%, about 90%,about 95% or more of the proteins in the composition.

Various methods for quantifying the degree of purification of theprotein or peptide will be known to those of skill in the art in lightof the present disclosure. These include, for example, determining thespecific activity of an active fraction, or assessing the amount ofpolypeptides within a fraction by SDS/PAGE analysis. A preferred methodfor assessing the purity of a fraction is to calculate the specificactivity of the fraction, to compare it to the specific activity of theinitial extract, and to thus calculate the degree of purity, hereinassessed by a “-fold purification number.” The actual units used torepresent the amount of activity will, of course, be dependent upon theparticular assay technique chosen to follow the purification and whetheror not the expressed protein or peptide exhibits a detectable activity.

Various techniques suitable for use in protein purification will be wellknown to those of skill in the art. These include, for example,precipitation with ammonium sulphate, PEG, antibodies and the like or byheat denaturation, followed by centrifugation; chromatography steps suchas ion exchange, gel filtration, reverse phase, hydroxylapatite andaffinity chromatography; isoelectric focusing; gel electrophoresis; andcombinations of such and other techniques. As is generally known in theart, it is believed that the order of conducting the variouspurification steps may be changed, or that certain steps may be omitted,and still result in a suitable method for the preparation of asubstantially purified protein or peptide.

There is no general requirement that the protein or peptide always beprovided in their most purified state. Indeed, it is contemplated thatless substantially purified products will have utility in certainembodiments. Partial purification may be accomplished by using fewerpurification steps in combination, or by utilizing different forms ofthe same general purification scheme. For example, it is appreciatedthat a cation-exchange column chromatography performed utilizing an HPLCapparatus will generally result in a greater “-fold” purification thanthe same technique utilizing a low pressure chromatography system.Methods exhibiting a lower degree of relative purification may haveadvantages in total recovery of protein product, or in maintaining theactivity of an expressed protein.

It is known that the migration of a polypeptide can vary, sometimessignificantly, with different conditions of SDS/PAGE (Capaldi et al.,1977). It will therefore be appreciated that under differingelectrophoresis conditions, the apparent molecular weights of purifiedor partially purified expression products may vary.

High Performance Liquid Chromatography (HPLC) is characterized by a veryrapid separation with extraordinary resolution of peaks. This isachieved by the use of very fine particles and high pressure to maintainan adequate flow rate. Separation can be accomplished in a matter ofminutes, or at most an hour. Moreover, only a very small volume of thesample is needed because the particles are so small and close-packedthat the void volume is a very small fraction of the bed volume. Also,the concentration of the sample need not be very great because the bandsare so narrow that there is very little dilution of the sample.

Gel chromatography, or molecular sieve chromatography, is a special typeof partition chromatography that is based on molecular size. The theorybehind gel chromatography is that the column, which is prepared withtiny particles of an inert substance that contain small pores, separateslarger molecules from smaller molecules as they pass through or aroundthe pores, depending on their size. As long as the material of which theparticles are made does not adsorb the molecules, the sole factordetermining rate of flow is the size. Hence, molecules are eluted fromthe column in decreasing size, so long as the shape is relativelyconstant. Gel chromatography is unsurpassed for separating molecules ofdifferent size because separation is independent of all other factorssuch as pH, ionic strength, temperature, etc. There also is virtually noadsorption, less zone spreading and the elution volume is related in asimple matter to molecular weight.

Affinity Chromatography is a chromatographic procedure that relies onthe specific affinity between a substance to be isolated and a moleculethat it can specifically bind to. This is a receptor-ligand typeinteraction. The column material is synthesized by covalently couplingone of the binding partners to an insoluble matrix. The column materialis then able to specifically adsorb the substance from the solution.Elution occurs by changing the conditions to those in which binding willnot occur (alter pH, ionic strength, temperature, etc.).

A particular type of affinity chromatography useful in the purificationof carbohydrate containing compounds is lectin affinity chromatography.Lectins are a class of substances that bind to a variety ofpolysaccharides and glycoproteins. Lectins are usually coupled toagarose by cyanogen bromide. Conconavalin A coupled to Sepharose was thefirst material of this sort to be used and has been widely used in theisolation of polysaccharides and glycoproteins other lectins that havebeen include lentil lectin, wheat germ agglutinin which has been usefulin the purification of N-acetyl glucosaminyl residues and Helix pomatialectin. Lectins themselves are purified using affinity chromatographywith carbohydrate ligands. Lactose has been used to purify lectins fromcastor bean and peanuts; maltose has been useful in extracting lectinsfrom lentils and jack bean; N-acetyl-D galactosamine is used forpurifying lectins from soybean; N-acetyl glucosaminyl binds to lectinsfrom wheat germ; D-galactosamine has been used in obtaining lectins fromclams and L-fuctose will bind to lectins from lotus.

The matrix should be a substance that itself does not adsorb moleculesto any significant extent and that has a broad range of chemical,physical and thermal stability. The ligand should be coupled in such away as to not affect its binding properties. The ligand should alsoprovide relatively tight binding. And it should be possible to elute thesubstance without destroying the sample or the ligand. One of the mostcommon forms of affinity chromatography is immunoaffinitychromatography. The generation of antibodies that would be suitable foruse in accord with the present invention is discussed below.

F. Synthesis

Because of their relatively small size, the peptides of the inventioncan also be synthesized in solution or on a solid support in accordancewith conventional techniques. Various automatic synthesizers arecommercially available and can be used in accordance with knownprotocols. See, for example, Stewart and Young (1984); Tam et al.(1983); Merrifield (1986); and Barany and Merrifield (1979), eachincorporated herein by reference. Short peptide sequences, or librariesof overlapping peptides, usually from about 6 up to about 35 to 50 aminoacids, which correspond to the selected regions described herein, can bereadily synthesized and then screened in screening assays designed toidentify reactive peptides. Alternatively, recombinant DNA technologymay be employed wherein a nucleotide sequence which encodes a peptide ofthe invention is inserted into an expression vector, transformed ortransfected into an appropriate host cell and cultivated underconditions suitable for expression.

G. Antigen Compositions

The present invention also provides for the use of Notch3 proteins orpeptides as antigens for the immunization of animals relating to theproduction of antibodies. It is envisioned that either Notch3, orportions thereof, will be coupled, bonded, bound, conjugated orchemically-linked to one or more agents via linkers, polylinkers orderivatized amino acids. This may be performed such that a bispecific ormultivalent composition or vaccine is produced. It is further envisionedthat the methods used in the preparation of these compositions will befamiliar to those of skill in the art and should be suitable foradministration to animals, i.e., pharmaceutically acceptable. Particularagents are the carriers are keyhole limpet hemocyannin (KLH) or bovineserum albumin (BSA).

III. Nucleic Acids

The present invention also provides, in another embodiment, genesencoding Notch3 or fragments (peptides) thereof. A gene for the humanNotch3 molecule has been identified. The present invention is notlimited in scope to this gene, however, as one of ordinary skill in thecould readily identify related homologs in various other species (e.g.,mouse, rat, rabbit, dog. monkey, gibbon, chimp, ape, baboon, cow, pig,horse, sheep, cat and other species).

In addition, it should be clear that the present invention is notlimited to the specific nucleic acids disclosed herein. As discussedbelow, a “Notch3 gene” may contain a variety of different bases and yetstill produce a corresponding polypeptide that is functionallyindistinguishable from, and in some cases structurally identical to, thehuman gene disclosed herein.

Similarly, any reference to a nucleic acid should be read asencompassing a host cell containing that nucleic acid and, in somecases, capable of expressing the product of that nucleic acid. Inaddition to therapeutic considerations, cells expressing nucleic acidsof the present invention may prove useful in the context of screeningfor agents that induce, repress, inhibit, augment, interfere with,block, abrogate, stimulate or enhance the function of Notch3.

A. Nucleic Acids Encoding Notch3

Nucleic acids according to the present invention may encode an entireNotch3 coding sequence (Accession No. U97669; SEQ ID NO:1), a domain ofNotch3 that expresses a tumor suppressing function, or any otherfragment of the Notch3 sequences set forth herein. The nucleic acid maybe derived from genomic DNA, i.e., cloned directly from the genome of aparticular organism. In preferred embodiments, however, the nucleic acidwould comprise complementary DNA (cDNA). Also contemplated is a cDNAplus a natural intron or an intron derived from another gene; suchengineered molecules are sometime referred to as “mini-genes.” At aminimum, these and other nucleic acids of the present invention may beused as molecular weight standards in, for example, gel electrophoresis.

The term “cDNA” is intended to refer to DNA prepared using messenger RNA(mRNA) as template. The advantage of using a cDNA, as opposed to genomicDNA or DNA polymerized from a genomic, non- or partially-processed RNAtemplate, is that the cDNA primarily contains coding sequences of thecorresponding protein. There may be times when the full or partialgenomic sequence is preferred, such as where the non-coding regions arerequired for optimal expression or where non-coding regions such asintrons are to be targeted in an antisense strategy.

It also is contemplated that a given Notch3 from a given species may berepresented by natural variants that have slightly different nucleicacid sequences but, nonetheless, encode the same protein (see Table 1,above).

As used in this application, the term “a nucleic acid encoding a Notch3”refers to a nucleic acid molecule that has been isolated free of totalcellular nucleic acid. In certain embodiments, the invention concerns anucleic acid sequence essentially as set forth in SEQ ID NO:2. The term“as set forth in SEQ ID NO:2” means that the nucleic acid sequencesubstantially corresponds to a portion of SEQ ID NO:2. The term“functionally equivalent codon” is used herein to refer to codons thatencode the same amino acid, such as the six codons for arginine orserine, and also refers to codons that encode biologically equivalentamino acids, as discussed in the following pages.

Allowing for the degeneracy of the genetic code, sequences that have atleast about 50%, usually at least about 60%, more usually about 70%,most usually about 80%, preferably at least about 90% and mostpreferably about 95% of nucleotides that are identical to thenucleotides of SEQ ID NO:2. Sequences that are essentially the same asthose set forth in SEQ ID NO:2 also may be functionally defined assequences that are capable of hybridizing to a nucleic acid segmentcontaining the complement of SEQ ID NO:2 under standard conditions.

The DNA segments of the present invention include those encodingbiologically functional equivalent Notch3 proteins and peptides, asdescribed above. Such sequences may arise as a consequence of codonredundancy and amino acid functional equivalency that are known to occurnaturally within nucleic acid sequences and the proteins thus encoded.Alternatively, functionally equivalent proteins or peptides may becreated via the application of recombinant DNA technology, in whichchanges in the protein structure may be engineered, based onconsiderations of the properties of the amino acids being exchanged.Changes designed by man may be introduced through the application ofsite-directed mutagenesis techniques or may be introduced randomly andscreened later for the desired function, as described below.

B. Oligonucleotide Probes and Primers

Naturally, the present invention also encompasses DNA segments that arecomplementary, or essentially complementary, to the sequence set forthin SEQ ID NO:2. Nucleic acid sequences that are “complementary” arethose that are capable of base-pairing according to the standardWatson-Crick complementary rules. As used herein, the term“complementary sequences” means nucleic acid sequences that aresubstantially complementary, as may be assessed by the same nucleotidecomparison set forth above, or as defined as being capable ofhybridizing to the nucleic acid segment of SEQ ID NO:2 under relativelystringent conditions such as those described herein. Such sequences mayencode the entire Notch3 protein or functional or non-functionalfragments thereof.

Alternatively, the hybridizing segments may be shorter oligonucleotides.Sequences of 17 bases long should occur only once in the human genomeand, therefore, suffice to specify a unique target sequence. Althoughshorter oligomers are easier to make and increase in vivo accessibility,numerous other factors are involved in determining the specificity ofhybridization. Both binding affinity and sequence specificity of anoligonucleotide to its complementary target increases with increasinglength. It is contemplated that exemplary oligonucleotides of 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90,95, 100, 110, 114 or more base pairs will be used, although others arecontemplated. Longer polynucleotides encoding 250, 500, 1000, 1212,1500, 2000, 2500, 3000 or longer are contemplated as well. Sucholigonucleotides will find use, for example, as probes in Southern andNorthern blots and as primers in amplification reactions.

Suitable hybridization conditions will be well known to those of skillin the art. In certain applications, for example, substitution of aminoacids by site-directed mutagenesis, it is appreciated that lowerstringency conditions are required. Under these conditions,hybridization may occur even though the sequences of probe and targetstrand are not perfectly complementary, but are mismatched at one ormore positions. Conditions may be rendered less stringent by increasingsalt concentration and decreasing temperature. For example, a mediumstringency condition could be provided by about 0.1 to 0.25 M NaCl attemperatures of about 37° C. to about 55° C., while a low stringencycondition could be provided by about 0.15 M to about 0.9 M salt, attemperatures ranging from about 20° C. to about 55° C. Thus,hybridization conditions can be readily manipulated, and thus willgenerally be a method of choice depending on the desired results.

In other embodiments, hybridization may be achieved under conditions of,for example, 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl₂, 10 mMdithiothreitol, at temperatures between approximately 20° C. to about37° C. Other hybridization conditions utilized could includeapproximately 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 μM MgCl₂, attemperatures ranging from approximately 40° C. to about 72° C. Formamideand SDS also may be used to alter the hybridization conditions.

One method of using probes and primers of the present invention is inthe search for genes related to Notch3 or, more particularly, homologsof Notch3 from other species. Normally, the target DNA will be a genomicor cDNA library, although screening may involve analysis of RNAmolecules. By varying the stringency of hybridization, and the region ofthe probe, different degrees of homology may be discovered.

Another way of exploiting probes and primers of the present invention isin site-directed, or site-specific mutagenesis. Site-specificmutagenesis is a technique useful in the preparation of individualpeptides, or biologically functional equivalent proteins or peptides,through specific mutagenesis of the underlying DNA. The techniquefurther provides a ready ability to prepare and test sequence variants,incorporating one or more of the foregoing considerations, byintroducing one or more nucleotide sequence changes into the DNA.Site-specific mutagenesis allows the production of mutants through theuse of specific oligonucleotide sequences which encode the DNA sequenceof the desired mutation, as well as a sufficient number of adjacentnucleotides, to provide a primer sequence of sufficient size andsequence complexity to form a stable duplex on both sides of thedeletion junction being traversed. Typically, a primer of about 17 to 25nucleotides in length is preferred, with about 5 to 10 residues on bothsides of the junction of the sequence being altered.

The technique typically employs a bacteriophage vector that exists inboth a single-stranded and double-stranded form. Typical vectors usefulin site-directed mutagenesis include vectors such as the M13 phage.These phage vectors are commercially available and their use isgenerally well known to those skilled in the art. Double-strandedplasmids are also routinely employed in site directed mutagenesis, whicheliminates the step of transferring the gene of interest from a phage toa plasmid.

In general, site-directed mutagenesis is performed by first obtaining asingle-stranded vector, or melting of two strands of a double-strandedvector which includes within its sequence a DNA sequence encoding thedesired protein. An oligonucleotide primer bearing the desired mutatedsequence is synthetically prepared. This primer is then annealed withthe single-stranded DNA preparation, taking into account the degree ofmismatch when selecting hybridization conditions, and subjected to DNApolymerizing enzymes such as E. coli polymerase I Klenow fragment, inorder to complete the synthesis of the mutation-bearing strand. Thus, aheteroduplex is formed wherein one strand encodes the originalnon-mutated sequence and the second strand bears the desired mutation.This heteroduplex vector is then used to transform appropriate cells,such as E. coli cells, and clones are selected that include recombinantvectors bearing the mutated sequence arrangement.

The preparation of sequence variants of the selected gene usingsite-directed mutagenesis is provided as a means of producingpotentially useful species and is not meant to be limiting, as there areother ways in which sequence variants of genes may be obtained. Forexample, recombinant vectors encoding the desired gene may be treatedwith mutagenic agents, such as hydroxylamine, to obtain sequencevariants.

C. Vectors for Cloning, Gene Transfer and Expression

Within certain embodiments, expression vectors are employed to expressthe Notch3 polypeptide product, which can then be purified for varioususes. In other embodiments, the expression vectors are used in genetherapy. Expression requires that appropriate signals be provided in thevectors, and which include various regulatory elements, such asenhancers/promoters from both viral and mammalian sources that driveexpression of the genes of interest in host cells. Elements designed tooptimize messenger RNA stability and translatability in host cells alsoare defined. The conditions for the use of a number of dominant drugselection markers for establishing permanent, stable cell clonesexpressing the products are also provided, as is an element that linksexpression of the drug selection markers to expression of thepolypeptide.

Throughout this application, the term “expression construct” is meant toinclude any type of genetic construct containing a nucleic acid codingfor a gene product in which part or all of the nucleic acid encodingsequence is capable of being transcribed. The transcript may betranslated into a protein, but it need not be. In certain embodiments,expression includes both transcription of a gene and translation of mRNAinto a gene product. In other embodiments, expression only includestranscription of the nucleic acid encoding a gene of interest.

The term “vector” is used to refer to a carrier nucleic acid moleculeinto which a nucleic acid sequence can be inserted for introduction intoa cell where it can be replicated. A nucleic acid sequence can be“exogenous,” which means that it is foreign to the cell into which thevector is being introduced or that the sequence is homologous to asequence in the cell but in a position within the host cell nucleic acidin which the sequence is ordinarily not found. Vectors include plasmids,cosmids, viruses (bacteriophage, animal viruses, and plant viruses), andartificial chromosomes (e.g., YACs). One of skill in the art would bewell equipped to construct a vector through standard recombinanttechniques, which are described in Sambrook et al. (1989) and Ausubel etal. (1994), both incorporated herein by reference.

The term “expression vector” refers to a vector containing a nucleicacid sequence coding for at least part of a gene product capable ofbeing transcribed. In some cases, RNA molecules are then translated intoa protein, polypeptide, or peptide. In other cases, these sequences arenot translated, for example, in the production of antisense molecules orribozymes. Expression vectors can contain a variety of “controlsequences,” which refer to nucleic acid sequences necessary for thetranscription and possibly translation of an operably linked codingsequence in a particular host organism. In addition to control sequencesthat govern transcription and translation, vectors and expressionvectors may contain nucleic acid sequences that serve other functions aswell and are described infra.

(i) Regulatory Elements

A “promoter” is a control sequence that is a region of a nucleic acidsequence at which initiation and rate of transcription are controlled.It may contain genetic elements at which regulatory proteins andmolecules may bind such as RNA polymerase and other transcriptionfactors. The phrases “operatively positioned,” “operatively linked,”“under control,” and “under transcriptional control” mean that apromoter is in a correct functional location and/or orientation inrelation to a nucleic acid sequence to control transcriptionalinitiation and/or expression of that sequence. A promoter may or may notbe used in conjunction with an “enhancer,” which refers to a cis-actingregulatory sequence involved in the transcriptional activation of anucleic acid sequence.

A promoter may be one naturally-associated with a gene or sequence, asmay be obtained by isolating the 5′ non-coding sequences locatedupstream of the coding segment and/or exon. Such a promoter can bereferred to as “endogenous.” Similarly, an enhancer may be one naturallyassociated with a nucleic acid sequence, located either downstream orupstream of that sequence. Alternatively, certain advantages will begained by positioning the coding nucleic acid segment under the controlof a recombinant or heterologous promoter, which refers to a promoterthat is not normally associated with a nucleic acid sequence in itsnatural environment. A recombinant or heterologous enhancer refers alsoto an enhancer not normally associated with a nucleic acid sequence inits natural environment. Such promoters or enhancers may includepromoters or enhancers of other genes, and promoters or enhancersisolated from any other prokaryotic, viral, or eukaryotic cell, andpromoters or enhancers not “naturally-occurring,” i.e., containingdifferent elements of different transcriptional regulatory regions,and/or mutations that alter expression. In addition to producing nucleicacid sequences of promoters and enhancers synthetically, sequences maybe produced using recombinant cloning and/or nucleic acid amplificationtechnology, including PCR™, in connection with the compositionsdisclosed herein (see U.S. Pat. No. 4,683,202, U.S. Pat. No. 5,928,906,each incorporated herein by reference). Furthermore, it is contemplatedthe control sequences that direct transcription and/or expression ofsequences within non-nuclear organelles such as mitochondria,chloroplasts, and the like, can be employed as well.

Naturally, it will be important to employ a promoter and/or enhancerthat effectively directs the expression of the DNA segment in the celltype, organelle, and organism chosen for expression. One example is thenative Notch3 promoter. Those of skill in the art of molecular biologygenerally know the use of promoters, enhancers, and cell typecombinations for protein expression, for example, see Sambrook et al.(1989), incorporated herein by reference. The promoters employed may beconstitutive, tissue-specific, inducible, and/or useful under theappropriate conditions to direct high level expression of the introducedDNA segment, such as is advantageous in the large-scale production ofrecombinant proteins and/or peptides. The promoter may be heterologousor endogenous.

Table 3 lists several elements/promoters that may be employed, in thecontext of the present invention, to regulate the expression of a gene.This list is not intended to be exhaustive of all the possible elementsinvolved in the promotion of expression but, merely, to be exemplarythereof. Table 4 provides examples of inducible elements, which areregions of a nucleic acid sequence that can be activated in response toa specific stimulus.

TABLE 3 Promoter and/or Enhancer Promoter/Enhancer ReferencesImmunoglobulin Heavy Chain Banerji et al., 1983; Gilles et al., 1983;Grosschedl et al., 1985; Atchinson et al., 1986, 1987; Imler et al.,1987; Weinberger et al., 1984; Kiledjian et al., 1988; Porton et al.;1990 Immunoglobulin Light Chain Queen et al., 1983; Picard et al., 1984T-Cell Receptor Luria et al., 1987; Winoto et al., 1989; Redondo et al.;1990 HLA DQ a and/or DQ β Sullivan et al., 1987 β-Interferon Goodbournet al., 1986; Fujita et al., 1987; Goodbourn et al., 1988 Interleukin-2Greene et al., 1989 Interleukin-2 Receptor Greene et al., 1989; Lin etal., 1990 MHC Class II 5 Koch et al., 1989 MHC Class II HLA-DRa Shermanet al., 1989 β-Actin Kawamoto et al., 1988; Ng et al.; 1989 MuscleCreatine Kinase (MCK) Jaynes et al., 1988; Horlick et al., 1989; Johnsonet al., 1989 Prealbumin (Transthyretin) Costa et al., 1988 Elastase IOrnitz et al., 1987 Metallothionein (MTII) Karin et al., 1987; Culottaet al., 1989 Collagenase Pinkert et al., 1987; Angel et al., 1987Albumin Pinkert et al., 1987; Tronche et al., 1989, 1990 α-FetoproteinGodbout et al., 1988; Campere et al., 1989 t-Globin Bodine et al., 1987;Perez-Stable et al., 1990 β-Globin Trudel et al., 1987 c-fos Cohen etal., 1987 c-HA-ras Triesman, 1986; Deschamps et al., 1985 Insulin Edlundet al., 1985 Neural Cell Adhesion Molecule Hirsh et al., 1990 (NCAM)α₁-Antitrypain Latimer et al., 1990 H2B (TH2B) Histone Hwang et al.,1990 Mouse and/or Type I Collagen Ripe et al., 1989 Glucose-RegulatedProteins Chang et al., 1989 (GRP94 and GRP78) Rat Growth Hormone Larsenet al., 1986 Human Serum Amyloid A (SAA) Edbrooke et al., 1989 TroponinI (TN I) Yutzey et al., 1989 Platelet-Derived Growth Factor Pech et al.,1989 (PDGF) Duchenne Muscular Dystrophy Klamut et al., 1990 SV40 Banerjiet al., 1981; Moreau et al., 1981; Sleigh et al., 1985; Firak et al.,1986; Herr et al., 1986; Imbra et al., 1986; Kadesch et al., 1986; Wanget al., 1986; Ondek et al., 1987; Kuhl et al., 1987; Schaffner et al.,1988 Polyoma Swartzendruber et al., 1975; Vasseur et al., 1980; Katinkaet al., 1980, 1981; Tyndell et al., 1981; Dandolo et al., 1983; deVilliers et al., 1984; Hen et al., 1986; Satake et al., 1988; Campbelland/or Villarreal, 1988 Retroviruses Kriegler et al., 1982, 1983;Levinson et al., 1982; Kriegler et al., 1983, 1984a, b, 1988; Bosze etal., 1986; Miksicek et al., 1986; Celander et al., 1987; Thiesen et al.,1988; Celander et al., 1988; Chol et al., 1988; Reisman et al., 1989Papilloma Virus Campo et al., 1983; Lusky et al., 1983; Spandidos and/orWilkie, 1983; Spalholz et al., 1985; Lusky et al., 1986; Cripe et al.,1987; Gloss et al., 1987; Hirochika et al., 1987; Stephens et al., 1987;Glue et al., 1988 Hepatitis B Virus Bulla et al., 1986; Jameel et al.,1986; Shaul et al., 1987; Spandau et al., 1988; Vannice et al., 1988Human Immunodeficiency Virus Muesing et al., 1987; Hauber et al., 1988;Jakobovits et al., 1988; Feng et al., 1988; Takebe et al., 1988; Rosenet al., 1988; Berkhout et al., 1989; Laspia et al., 1989; Sharp et al.,1989; Braddock et al., 1989 Cytomegalovirus (CMV) Weber et al., 1984;Boshart et al., 1985; Foecking et al., 1986 Gibbon Ape Leukemia VirusHolbrook et al., 1987; Quinn et al., 1989

TABLE 4 Inducible Elements Element Inducer References MT II PhorbolEster (TFA) Palmiter et al., 1982; Haslinger et Heavy metals al., 1985;Searle et al., 1985; Stuart et al., 1985; Imagawa et al., 1987, Karin etal., 1987; Angel et al., 1987b; McNeall et al., 1989 MMTV (mouse mammaryGlucocorticoids Huang et al., 1981; Lee et al., tumor virus) 1981;Majors et al., 1983; Chandler et al., 1983; Lee et al., 1984; Ponta etal., 1985; Sakai et al., 1988 β-Interferon poly(rI) × Tavernier et al.,1983 poly(rc) Adenovirus 5 E2 ElA Imperiale et al., 1984 CollagenasePhorbol Ester (TPA) Angel et al., 1987a Stromelysin Phorbol Ester (TPA)Angel et al., 1987b SV40 Phorbol Ester (TPA) Angel et al., 1987b MurineMX Gene Interferon, Newcastle Hug et al., 1988 Disease Virus GRP78 GeneA23187 Resendez et al., 1988 α-2-Macroglobulin IL-6 Kunz et al., 1989Vimentin Serum Rittling et al., 1989 MHC Class I Gene H-2κb InterferonBlanar et al., 1989 HSP70 ElA, SV40 Large T Taylor et al., 1989, 1990a,1990b Antigen Proliferin Phorbol Ester-TPA Mordacq et al., 1989 TumorNecrosis Factor PMA Hensel et al., 1989 Thyroid Stimulating ThyroidHormone Chatterjee et al., 1989 Hormone α Gene

The identity of tissue-specific promoters or elements, as well as assaysto characterize their activity, is well known to those of skill in theart. Examples of such regions include the human LIMK2 gene (Nomoto etal. 1999), the somatostatin receptor 2 gene (Kraus et al., 1998), murineepididymal retinoic acid-binding gene (Lareyre et al., 1999), human CD4(Zhao-Emonet et al., 1998), mouse alpha2 (XI) collagen (Tsumaki, et al.,1998), D1A dopamine receptor gene (Lee, et al., 1997), insulin-likegrowth factor II (Wu et al., 1997), human platelet endothelial celladhesion molecule-1 (Almendro et al., 1996). Tumor specific promotersalso will find use in the present invention. Some such promoters are setforth in Tables 4 and 5.

TABLE 5 Candidate Tissue-Specific Promoters for Cancer Gene TherapyCancers in which promoter Normal cells in which Tissue-specific promoteris active promoter is active Carcinoembryonic antigen Most colorectalcarcinomas; Colonic mucosa; gastric (CEA)* 50% of lung carcinomas;40-50% mucosa; lung epithelia; of gastric carcinomas; eccrine sweatglands; cells in most pancreatic carcinomas; testes many breastcarcinomas Prostate-specific antigen Most prostate carcinomas Prostateepithelium (PSA) Vasoactive intestinal peptide Majority of non-smallcell Neurons; lymphocytes; mast (VIP) lung cancers cells; eosinophilsSurfactant protein A (SP-A) Many lung adenocarcinomas Type IIpneumocytes; Clara cells Human achaete-scute Most small cell lungcancers Neuroendocrine cells in lung homolog (hASH) Mucin-1 (MUC1)**Most adenocarcinomas Glandular epithelial cells in (originating from anytissue) breast and in respiratory, gastrointestinal, and genitourinarytracts Alpha-fetoprotein Most hepatocellular Hepatocytes (under certaincarcinomas; possibly many conditions); testis testicular cancers AlbuminMost hepatocellular Hepatocytes carcinomas Tyrosinase Most melanomasMelanocytes; astrocytes; Schwann cells; some neurons Tyrosine-bindingprotein Most melanomas Melanocytes; astrocytes, (TRP) Schwann cells;some neurons Keratin 14 Presumably many squamous Keratinocytes cellcarcinomas (e.g., Head and neck cancers) EBV LD-2 Many squamous cellKeratinocytes of upper carcinomas of head and neck digestiveKeratinocytes of upper digestive tract Glial fibrillary acidic proteinMany astrocytomas Astrocytes (GFAP) Myelin basic protein (MBP) Manygliomas Oligodendrocytes Testis-specific angiotensin- Possibly manytesticular Spermatazoa converting enzyme (Testis- cancers specific ACE)Osteocalcin Possibly many osteosarcomas Osteoblasts

TABLE 6 Candidate Promoters for Tissue-Specific Targeting of TumorsCancers in which Promoter Normal cells in which Promoter is activePromoter is active E2F-regulated promoter Almost all cancersProliferating cells HLA-G Many colorectal carcinomas; Lymphocytes;monocytes; many melanomas; possibly spermatocytes; trophoblast manyother cancers FasL Most melanomas; many Activated leukocytes: pancreaticcarcinomas; most neurons; endothelial cells; astrocytomas possibly manykeratinocytes; cells in other cancers immunoprivileged tissues; somecells in lungs, ovaries, liver, and prostate Myc-regulated promoter Mostlung carcinomas (both Proliferating cells (only some small cell andnon-small cell); cell-types): mammary most colorectal carcinomasepithelial cells (including non- proliferating) MAGE-1 Many melanomas;some non- Testis small cell lung carcinomas; some breast carcinomas VEGF70% of all cancers Cells at sites of (constitutive overexpression inneovascularization (but unlike many cancers) in tumors, expression istransient, less strong, and never constitutive) bFGF Presumably manydifferent Cells at sites of ischemia (but cancers, since bFGF unliketumors, expression is expression is induced by transient, less strong,and ischemic conditions never constitutive) COX-2 Most colorectalcarcinomas; Cells at sites of inflammation many lung carcinomas;possibly many other cancers IL-10 Most colorectal carcinomas; Leukocytesmany lung carcinomas; many squamous cell carcinomas of head and neck;possibly many other cancers GRP78/BiP Presumably many different Cells atsites of ishemia cancers, since GRP7S expression is induced bytumor-specific conditions CarG elements from Egr-1 Induced by ionizationCells exposed to ionizing radiation, so conceivably most radiation;leukocytes tumors upon irradiation

A specific initiation signal also may be required for efficienttranslation of coding sequences. These signals include the ATGinitiation codon or adjacent sequences. Exogenous translational controlsignals, including the ATG initiation codon, may need to be provided.One of ordinary skill in the art would readily be capable of determiningthis and providing the necessary signals. It is well known that theinitiation codon must be “in-frame” with the reading frame of thedesired coding sequence to ensure translation of the entire insert. Theexogenous translational control signals and initiation codons can beeither natural or synthetic. The efficiency of expression may beenhanced by the inclusion of appropriate transcription enhancerelements.

(ii) IRES

In certain embodiments of the invention, the use of internal ribosomeentry sites (IRES) elements are used to create multigene, orpolycistronic, messages. IRES elements are able to bypass the ribosomescanning model of 5′-methylated Cap dependent translation and begintranslation at internal sites (Pelletier and Sonenberg, 1988). IRESelements from two members of the picornavirus family (polio andencephalomyocarditis) have been described (Pelletier and Sonenberg,1988), as well an IRES from a mammalian message (Macejak and Sarnow,1991). IRES elements can be linked to heterologous open reading frames.Multiple open reading frames can be transcribed together, each separatedby an IRES, creating polycistronic messages. By virtue of the IRESelement, each open reading frame is accessible to ribosomes forefficient translation. Multiple genes can be efficiently expressed usinga single promoter/enhancer to transcribe a single message (see U.S. Pat.Nos. 5,925,565 and 5,935,819, herein incorporated by reference).

(iii) Multi-Purpose Cloning Sites

Vectors can include a multiple cloning site (MCS), which is a nucleicacid region that contains multiple restriction enzyme sites, any ofwhich can be used in conjunction with standard recombinant technology todigest the vector. See Carbonelli et al., 1999, Levenson et al., 1998,and Cocea, 1997, incorporated herein by reference. “Restriction enzymedigestion” refers to catalytic cleavage of a nucleic acid molecule withan enzyme that functions only at specific locations in a nucleic acidmolecule. Many of these restriction enzymes are commercially available.Use of such enzymes is widely understood by those of skill in the art.Frequently, a vector is linearized or fragmented using a restrictionenzyme that cuts within the MCS to enable exogenous sequences to beligated to the vector. “Ligation” refers to the process of formingphosphodiester bonds between two nucleic acid fragments, which may ormay not be contiguous with each other. Techniques involving restrictionenzymes and ligation reactions are well known to those of skill in theart of recombinant technology.

(iv) Splicing Sites

Most transcribed eukaryotic RNA molecules will undergo RNA splicing toremove introns from the primary transcripts. Vectors containing genomiceukaryotic sequences may require donor and/or acceptor splicing sites toensure proper processing of the transcript for protein expression (seeChandler et al., 1997, herein incorporated by reference.)

(v) Termination Signals

The vectors or constructs of the present invention will generallycomprise at least one termination signal. A “termination signal” or“terminator” is comprised of the DNA sequences involved in specifictermination of an RNA transcript by an RNA polymerase. Thus, in certainembodiments a termination signal that ends the production of an RNAtranscript is contemplated. A terminator may be necessary in vivo toachieve desirable message levels.

In eukaryotic systems, the terminator region may also comprise specificDNA sequences that permit site-specific cleavage of the new transcriptso as to expose a polyadenylation site. This signals a specializedendogenous polymerase to add a stretch of about 200 A residues (polyA)to the 3′ end of the transcript. RNA molecules modified with this polyAtail appear to more stable and are translated more efficiently. Thus, inother embodiments involving eukaryotes, it is preferred that thatterminator comprises a signal for the cleavage of the RNA, and it ismore preferred that the terminator signal promotes polyadenylation ofthe message. The terminator and/or polyadenylation site elements canserve to enhance message levels and/or to minimize read through from thecassette into other sequences.

Terminators contemplated for use in the invention include any knownterminator of transcription described herein or known to one of ordinaryskill in the art, including but not limited to, for example, thetermination sequences of genes, such as for example the bovine growthhormone terminator or viral termination sequences, such as for examplethe SV40 terminator. In certain embodiments, the termination signal maybe a lack of transcribable or translatable sequence, such as due to asequence truncation.

(vi) Polyadenylation Signals

In expression, particularly eukaryotic expression, one will typicallyinclude a polyadenylation signal to effect proper polyadenylation of thetranscript. The nature of the polyadenylation signal is not believed tobe crucial to the successful practice of the invention, and/or any suchsequence may be employed. Preferred embodiments include the SV40polyadenylation signal and/or the bovine growth hormone polyadenylationsignal, convenient and/or known to function well in various targetcells. Polyadenylation may increase the stability of the transcript ormay facilitate cytoplasmic transport.

(vii) Origins of Replication

In order to propagate a vector in a host cell, it may contain one ormore origins of replication sites (often termed “ori”), which is aspecific nucleic acid sequence at which replication is initiated.Alternatively an autonomously replicating sequence (ARS) can be employedif the host cell is yeast.

(viii) Selectable and Screenable Markers

In certain embodiments of the invention, cells containing a nucleic acidconstruct of the present invention may be identified in vitro or in vivoby including a marker in the expression vector. Such markers wouldconfer an identifiable change to the cell permitting easy identificationof cells containing the expression vector. Generally, a selectablemarker is one that confers a property that allows for selection. Apositive selectable marker is one in which the presence of the markerallows for its selection, while a negative selectable marker is one inwhich its presence prevents its selection. An example of a positiveselectable marker is a drug resistance marker.

Usually the inclusion of a drug selection marker aids in the cloning andidentification of transformants, for example, genes that conferresistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin andhistidinol are useful selectable markers. In addition to markersconferring a phenotype that allows for the discrimination oftransformants based on the implementation of conditions, other types ofmarkers including screenable markers such as GFP, whose basis iscalorimetric analysis, are also contemplated. Alternatively, screenableenzymes such as herpes simplex virus thymidine kinase (tk) orchloramphenicol acetyltransferase (CAT) may be utilized. One of skill inthe art would also know how to employ immunologic markers, possibly inconjunction with FACS analysis. The marker used is not believed to beimportant, so long as it is capable of being expressed simultaneouslywith the nucleic acid encoding a gene product. Further examples ofselectable and screenable markers are well known to one of skill in theart.

(ix) Viral Vectors

The capacity of certain viral vectors to efficiently infect or entercells, to integrate into a host cell genome and stably express viralgenes, have led to the development and application of a number ofdifferent viral vector systems (Robbins et al., 1998). Viral systems arecurrently being developed for use as vectors for ex vivo and in vivogene transfer. For example, adenovirus, herpes-simplex virus, retrovirusand adeno-associated virus vectors are being evaluated currently fortreatment of diseases such as cancer, cystic fibrosis, Gaucher disease,renal disease and arthritis (Robbins and Ghivizzani, 1998; Imai et al.,1998; U.S. Pat. No. 5,670,488). The various viral vectors describedbelow, present specific advantages and disadvantages, depending on theparticular gene-therapeutic application.

Adenoviral Vectors. In particular embodiments, an adenoviral expressionvector is contemplated for the delivery of expression constructs.“Adenovirus expression vector” is meant to include those constructscontaining adenovirus sequences sufficient to (a) support packaging ofthe construct and (b) to ultimately express a tissue or cell-specificconstruct that has been cloned therein.

Adenoviruses comprise linear, double-stranded DNA, with a genome rangingfrom 30 to 35 kb in size (Reddy et al., 1998; Morrison et al., 1997;Chillon et al., 1999). An adenovirus expression vector according to thepresent invention comprises a genetically engineered form of theadenovirus. Advantages of adenoviral gene transfer include the abilityto infect a wide variety of cell types, including non-dividing cells, amid-sized genome, ease of manipulation, high infectivity and the abilityto be grown to high titers (Wilson, 1996). Further, adenoviral infectionof host cells does not result in chromosomal integration becauseadenoviral DNA can replicate in an episomal manner, without potentialgenotoxicity associated with other viral vectors. Adenoviruses also arestructurally stable (Marienfeld et al., 1999) and no genomerearrangement has been detected after extensive amplification (Parks etal., 1997; Bett et al., 1993).

Salient features of the adenovirus genome are an early region (E1, E2,E3 and E4 genes), an intermediate region (pIX gene, Iva2 gene), a lateregion (L1, L2, L3, L4 and L5 genes), a major late promoter (MLP),inverted-terminal-repeats (ITRs) and a ψ sequence (Zheng, et al., 1999;Robbins et al., 1998; Graham and Prevec, 1995). The early genes E1, E2,E3 and E4 are expressed from the virus after infection and encodepolypeptides that regulate viral gene expression, cellular geneexpression, viral replication, and inhibition of cellular apoptosis.Further on during viral infection, the MLP is activated, resulting inthe expression of the late (L) genes, encoding polypeptides required foradenovirus encapsidation. The intermediate region encodes components ofthe adenoviral capsid. Adenoviral inverted terminal repeats (ITRs;100-200 bp in length), are cis elements, and function as origins ofreplication and are necessary for viral DNA replication. The ψ sequenceis required for the packaging of the adenoviral genome.

A common approach for generating an adenoviruses for use as a genetransfer vector is the deletion of the E1 gene (E1⁻), which is involvedin the induction of the E2, E3 and E4 promoters (Graham and Prevec,1995). Subsequently, a therapeutic gene or genes can be insertedrecombinantly in place of the E1 gene, wherein expression of thetherapeutic gene(s) is driven by the E1 promoter or a heterologouspromoter. The E1⁻, replication-deficient virus is then proliferated in a“helper” cell line that provides the E1 polypeptides in trans (e.g., thehuman embryonic kidney cell line 293). Thus, in the present invention itmay be convenient to introduce the transforming construct at theposition from which the E1-coding sequences have been removed. However,the position of insertion of the construct within the adenovirussequences is not critical to the invention. Alternatively, the E3region, portions of the E4 region or both may be deleted, wherein aheterologous nucleic acid sequence under the control of a promoteroperable in eukaryotic cells is inserted into the adenovirus genome foruse in gene transfer (U.S. Pat. No. 5,670,488; U.S. Pat. No. 5,932,210,each specifically incorporated herein by reference).

Although adenovirus based vectors offer several unique advantages overother vector systems, they often are limited by vector immunogenicity,size constraints for insertion of recombinant genes and low levels ofreplication. The preparation of a recombinant adenovirus vector deletedof all open reading frames, comprising a full length dystrophin gene andthe terminal repeats required for replication (Haecker et al., 1996)offers some potentially promising advantages to the above mentionedadenoviral shortcomings. The vector was grown to high titer with ahelper virus in 293 cells and was capable of efficiently transducingdystrophin in mdx mice, in myotubes in vitro and muscle fibers in vivo.Helper-dependent viral vectors are discussed below.

A major concern in using adenoviral vectors is the generation of areplication-competent virus during vector production in a packaging cellline or during gene therapy treatment of an individual. The generationof a replication-competent virus could pose serious threat of anunintended viral infection and pathological consequences for thepatient. Armentano et al. (1990), describe the preparation of areplication-defective adenovirus vector, claimed to eliminate thepotential for the inadvertent generation of a replication-competentadenovirus (U.S. Pat. No. 5,824,544, specifically incorporated herein byreference). The replication-defective adenovirus method comprises adeleted E1 region and a relocated protein IX gene, wherein the vectorexpresses a heterologous, mammalian gene.

Other than the requirement that the adenovirus vector be replicationdefective, or at least conditionally defective, the nature of theadenovirus vector is not believed to be crucial to the successfulpractice of the invention. The adenovirus may be of any of the 42different known serotypes and/or subgroups A-F. Adenovirus type 5 ofsubgroup C is the preferred starting material in order to obtain theconditional replication-defective adenovirus vector for use in thepresent invention. This is because adenovirus type 5 is a humanadenovirus about which a great deal of biochemical and geneticinformation is known, and it has historically been used for mostconstructions employing adenovirus as a vector.

As stated above, the typical vector according to the present inventionis replication defective and will not have an adenovirus E1 region.Adenovirus growth and manipulation is known to those of skill in theart, and exhibits broad host range in vitro and in vivo (U.S. Pat. No.5,670,488; U.S. Pat. No. 5,932,210; U.S. Pat. No. 5,824,544). This groupof viruses can be obtained in high titers, e.g., 10⁹ to 10¹¹plaque-forming units per ml, and they are highly infective. The lifecycle of adenovirus does not require integration into the host cellgenome. The foreign genes delivered by adenovirus vectors are episomaland, therefore, have low genotoxicity to host cells. Many experiments,innovations, preclinical studies and clinical trials are currently underinvestigation for the use of adenoviruses as gene delivery vectors. Forexample, adenoviral gene delivery-based gene therapies are beingdeveloped for liver diseases (Han et al., 1999), psychiatric diseases(Lesch, 1999), neurological diseases (Smith, 1998; Hermens andVerhaagen, 1998), coronary diseases (Feldman et al., 1996), musculardiseases (Petrof, 1998), gastrointestinal diseases (Wu, 1998) andvarious cancers such as colorectal (Fujiwara and Tanaka, 1998; Dorai etal., 1999), pancreatic, bladder (Irie et al., 1999), head and neck(Blackwell et al., 1999), breast (Stewart et al., 1999), lung (Batra etal., 1999) and ovarian (Vanderkwaak et al., 1999).

Retroviral Vectors. In certain embodiments of the invention, the use ofretroviruses for gene delivery are contemplated. Retroviruses are RNAviruses comprising an RNA genome. When a host cell is infected by aretrovirus, the genomic RNA is reverse transcribed into a DNAintermediate which is integrated into the chromosomal DNA of infectedcells. This integrated DNA intermediate is referred to as a provirus. Aparticular advantage of retroviruses is that they can stably infectdividing cells with a gene of interest (e.g., a therapeutic gene) byintegrating into the host DNA, without expressing immunogenic viralproteins. Theoretically, the integrated retroviral vector will bemaintained for the life of the infected host cell, expressing the geneof interest.

The retroviral genome and the proviral DNA have three genes: gag, pol,and env, which are flanked by two long terminal repeat (LTR) sequences.The gag gene encodes the internal structural (matrix, capsid, andnucleocapsid) proteins; the pol gene encodes the RNA-directed DNApolymerase (reverse transcriptase) and the env gene encodes viralenvelope glycoproteins. The 5′ and 3′ LTRs serve to promotetranscription and polyadenylation of the virion RNAs. The LTR containsall other cis-acting sequences necessary for viral replication.

A recombinant retrovirus of the present invention may be geneticallymodified in such a way that some of the structural, infectious genes ofthe native virus have been removed and replaced instead with a nucleicacid sequence to be delivered to a target cell (U.S. Pat. No. 5,858,744;U.S. Pat. No. 5,739,018, each incorporated herein by reference). Afterinfection of a cell by the virus, the virus injects its nucleic acidinto the cell and the retrovirus genetic material can integrate into thehost cell genome. The transferred retrovirus genetic material is thentranscribed and translated into proteins within the host cell. As withother viral vector systems, the generation of a replication-competentretrovirus during vector production or during therapy is a majorconcern. Retroviral vectors suitable for use in the present inventionare generally defective retroviral vectors that are capable of infectingthe target cell, reverse transcribing their RNA genomes, and integratingthe reverse transcribed DNA into the target cell genome, but areincapable of replicating within the target cell to produce infectiousretroviral particles (e.g., the retroviral genome transferred into thetarget cell is defective in gag, the gene encoding virion structuralproteins, and/or in pol, the gene encoding reverse transcriptase). Thus,transcription of the provirus and assembly into infectious virus occursin the presence of an appropriate helper virus or in a cell linecontaining appropriate sequences enabling encapsidation withoutcoincident production of a contaminating helper virus.

The growth and maintenance of retroviruses is known in the art (U.S.Pat. No. 5,955,331; U.S. Pat. No. 5,888,502, each specificallyincorporated herein by reference). Nolan et al. describe the productionof stable high titre, helper-free retrovirus comprising a heterologousgene (U.S. Pat. No. 5,830,725, specifically incorporated herein byreference). Methods for constructing packaging cell lines useful for thegeneration of helper-free recombinant retroviruses with amphoteric orecotrophic host ranges, as well as methods of using the recombinantretroviruses to introduce a gene of interest into eukaryotic cells invivo and in vitro are contemplated in the present invention (U.S. Pat.No. 5,955,331).

Currently, the majority of all clinical trials for vector-mediated genedelivery use murine leukemia virus (MLV)-based retroviral vector genedelivery (Robbins et al., 1998; Miller et al., 1993). Disadvantages ofretroviral gene delivery includes a requirement for ongoing celldivision for stable infection and a coding capacity that prevents thedelivery of large genes. However, recent development of vectors such aslentivirus (e.g., HIV), simian immunodeficiency virus (SIV) and equineinfectious-anemia virus (EIAV), which can infect certain non-dividingcells, potentially allow the in vivo use of retroviral vectors for genetherapy applications (Amado and Chen, 1999; Klimatcheva et al., 1999;White et al., 1999; Case et al., 1999). For example, HIV-based vectorshave been used to infect non-dividing cells such as neurons (Miyatake etal., 1999), islets (Leibowitz et al., 1999) and muscle cells (Johnstonet al., 1999). The therapeutic delivery of genes via retroviruses arecurrently being assessed for the treatment of various disorders such asinflammatory disease (Moldawer et al., 1999), AIDS (Amado and Chen,1999; Engel and Kohn, 1999), cancer (Clay et al., 1999), cerebrovasculardisease (Weihl et al., 1999) and hemophilia (Kay, 1998).

Herpesviral Vectors. Herpes simplex virus (HSV) type I and type IIcontain a double-stranded, linear DNA genome of approximately 150 kb,encoding 70-80 genes. Wild type HSV are able to infect cells lyticallyand to establish latency in certain cell types (e.g., neurons). Similarto adenovirus, HSV also can infect a variety of cell types includingmuscle (Yeung et al., 1999), ear (Derby et al., 1999), eye (Kaufman etal., 1999), tumors (Yoon et al., 1999; Howard et al., 1999), lung (Kohutet al., 1998), neuronal (Gamido et al., 1999; Lachmann and Efstathiou,1999), liver (Miyatake et al., 1999; Kooby et al., 1999) and pancreaticislets (Rabinovitch et al., 1999).

HSV viral genes are transcribed by cellular RNA polymerase II and aretemporally regulated, resulting in the transcription and subsequentsynthesis of gene products in roughly three discernable phases orkinetic classes. These phases of genes are referred to as the ImmediateEarly (IE) or alpha genes, Early (E) or beta genes and Late (L) or gammagenes. Immediately following the arrival of the genome of a virus in thenucleus of a newly infected cell, the IE genes are transcribed. Theefficient expression of these genes does not require prior viral proteinsynthesis. The products of IE genes are required to activatetranscription and regulate the remainder of the viral genome.

For use in therapeutic gene delivery, HSV must be renderedreplication-defective. Protocols for generating replication-defectiveHSV helper virus-free cell lines have been described (U.S. Pat. No.5,879,934; U.S. Pat. No. 5,851,826, each specifically incorporatedherein by reference in its entirety). One IE protein, Infected CellPolypeptide 4 (ICP4), also known as alpha 4 or Vmw175, is absolutelyrequired for both virus infectivity and the transition from IE to latertranscription. Thus, due to its complex, multifunctional nature andcentral role in the regulation of HSV gene expression, ICP4 hastypically been the target of HSV genetic studies.

Phenotypic studies of HSV viruses deleted of ICP4 indicate that suchviruses will be potentially useful for gene transfer purposes (Krisky etal., 1998a). One property of viruses deleted for ICP4 that makes themdesirable for gene transfer is that they only express the five other IEgenes: ICP0, ICP6, ICP27, ICP22 and ICP47 (DeLuca et al., 1985), withoutthe expression of viral genes encoding proteins that direct viral DNAsynthesis, as well as the structural proteins of the virus. Thisproperty is desirable for minimizing possible deleterious effects onhost cell metabolism or an immune response following gene transfer.Further deletion of IE genes ICP22 and ICP27, in addition to ICP4,substantially improve reduction of HSV cytotoxicity and prevented earlyand late viral gene expression (Krisky et al., 1998b).

The therapeutic potential of HSV in gene transfer has been demonstratedin various in vitro model systems and in vivo for diseases such asParkinson's (Yamada et al., 1999), retinoblastoma (Hayashi et al.,1999), intracerebral and intradermal tumors (Moriuchi et al., 1998),B-cell malignancies (Suzuki et al., 1998), ovarian cancer (Wang et al.,1998) and Duchenne muscular dystrophy (Huard et al., 1997).

Adeno-Associated Viral Vectors. Adeno-associated virus (AAV), a memberof the parvovirus family, is a human virus that is increasingly beingused for gene delivery therapeutics. AAV has several advantageousfeatures not found in other viral systems. First, AAV can infect a widerange of host cells, including non-dividing cells. Second, AAV caninfect cells from different species. Third, AAV has not been associatedwith any human or animal disease and does not appear to alter thebiological properties of the host cell upon integration. For example, itis estimated that 80-85% of the human population has been exposed toAAV. Finally, AAV is stable at a wide range of physical and chemicalconditions which lends itself to production, storage and transportationrequirements.

The AAV genome is a linear, single-stranded DNA molecule containing 4681nucleotides. The AAV genome generally comprises an internalnon-repeating genome flanked on each end by inverted terminal repeats(ITRs) of approximately 145 bp in length. The ITRs have multiplefunctions, including origins of DNA replication, and as packagingsignals for the viral genome. The internal non-repeated portion of thegenome includes two large open reading frames, known as the AAVreplication (rep) and capsid (cap) genes. The rep and cap genes code forviral proteins that allow the virus to replicate and package the viralgenome into a virion. A family of at least four viral proteins areexpressed from the AAV rep region, Rep 78, Rep 68, Rep 52, and Rep 40,named according to their apparent molecular weight. The AAV cap regionencodes at least three proteins, VP1, VP2, and VP3.

AAV is a helper-dependent virus requiring co-infection with a helpervirus (e.g., adenovirus, herpesvirus or vaccinia) in order to form AAVvirions. In the absence of co-infection with a helper virus, AAVestablishes a latent state in which the viral genome inserts into a hostcell chromosome, but infectious virions are not produced. Subsequentinfection by a helper virus “rescues” the integrated genome, allowing itto replicate and package its genome into infectious AAV virions.Although AAV can infect cells from different species, the helper virusmust be of the same species as the host cell (e.g., human AAV willreplicate in canine cells co-infected with a canine adenovirus).

AAV has been engineered to deliver genes of interest by deleting theinternal non-repeating portion of the AAV genome and inserting aheterologous gene between the ITRs. The heterologous gene may befunctionally linked to a heterologous promoter (constitutive,cell-specific, or inducible) capable of driving gene expression intarget cells. To produce infectious recombinant AAV (rAAV) containing aheterologous gene, a suitable producer cell line is transfected with arAAV vector containing a heterologous gene. The producer cell isconcurrently transfected with a second plasmid harboring the AAV rep andcap genes under the control of their respective endogenous promoters orheterologous promoters. Finally, the producer cell is infected with ahelper virus.

Once these factors come together, the heterologous gene is replicatedand packaged as though it were a wild-type AAV genome. When target cellsare infected with the resulting rAAV virions, the heterologous geneenters and is expressed in the target cells. Because the target cellslack the rep and cap genes and the adenovirus helper genes, the rAAVcannot further replicate, package or form wild-type AAV.

The use of helper virus, however, presents a number of problems. First,the use of adenovirus in a rAAV production system causes the host cellsto produce both rAAV and infectious adenovirus. The contaminatinginfectious adenovirus can be inactivated by heat treatment (56° C. for 1hour). Heat treatment, however, results in approximately a 50% drop inthe titer of functional rAAV virions. Second, varying amounts ofadenovirus proteins are present in these preparations. For example,approximately 50% or greater of the total protein obtained in such rAAVvirion preparations is free adenovirus fiber protein. If not completelyremoved, these adenovirus proteins have the potential of eliciting animmune response from the patient. Third, AAV vector production methodswhich employ a helper virus require the use and manipulation of largeamounts of high titer infectious helper virus, which presents a numberof health and safety concerns, particularly in regard to the use of aherpesvirus. Fourth, concomitant production of helper virus particles inrAAV virion producing cells diverts large amounts of host cellularresources away from rAAV virion production, potentially resulting inlower rAAV virion yields.

Lentiviral Vectors. Lentiviruses are complex retroviruses, which, inaddition to the common retroviral genes gag, pol, and env, contain othergenes with regulatory or structural function. The higher complexityenables the virus to modulate its life cycle, as in the course of latentinfection. Some examples of lentivirus include the HumanImmunodeficiency Viruses: HIV-1, HIV-2 and the Simian ImmunodeficiencyVirus: SIV. Lentiviral vectors have been generated by multiplyattenuating the HIV virulence genes, for example, the genes env, vif,vpr, vpu and nef are deleted making the vector biologically safe.

Recombinant lentiviral vectors are capable of infecting non-dividingcells and can be used for both in vivo and ex vivo gene transfer andexpression of nucleic acid sequences. The lentiviral genome and theproviral DNA have the three genes found in retroviruses: gag, pol andenv, which are flanked by two long terminal repeat (LTR) sequences. Thegag gene encodes the internal structural (matrix, capsid andnucleocapsid) proteins; the pol gene encodes the RNA-directed DNApolymerase (reverse transcriptase), a protease and an integrase; and theenv gene encodes viral envelope glycoproteins. The 5′ and 3′ LTR's serveto promote transcription and polyadenylation of the virion RNA's. TheLTR contains all other cis-acting sequences necessary for viralreplication. Lentiviruses have additional genes including vif, vpr, tat,rev, vpu, nef and vpx.

Adjacent to the 5′ LTR are sequences necessary for reverse transcriptionof the genome (the tRNA primer binding site) and for efficientencapsidation of viral RNA into particles (the Psi site). If thesequences necessary for encapsidation (or packaging of retroviral RNAinto infectious virions) are missing from the viral genome, the cisdefect prevents encapsidation of genomic RNA. However, the resultingmutant remains capable of directing the synthesis of all virionproteins.

Lentiviral vectors are known in the art, see Naldini et al., (1996);Zufferey et al., (1997); U.S. Pat. Nos. 6,013,516; and 5,994,136. Ingeneral, the vectors are plasmid-based or virus-based, and areconfigured to carry the essential sequences for incorporating foreignnucleic acid, for selection and for transfer of the nucleic acid into ahost cell. The gag, pol and env genes of the vectors of interest alsoare known in the art. Thus, the relevant genes are cloned into theselected vector and then used to transform the target cell of interest.

Recombinant lentivirus capable of infecting a non-dividing cell whereina suitable host cell is transfected with two or more vectors carryingthe packaging functions, namely gag, pol and env, as well as rev and tatis described in U.S. Pat. No. 5,994,136, incorporated herein byreference. This describes a first vector that can provide a nucleic acidencoding a viral gag and a pol gene and another vector that can providea nucleic acid encoding a viral env to produce a packaging cell.Introducing a vector providing a heterologous gene, such as the STAT-1αgene in this invention, into that packaging cell yields a producer cellwhich releases infectious viral particles carrying the foreign gene ofinterest. The env preferably is an amphotropic envelope protein whichallows transduction of cells of human and other species.

One may target the recombinant virus by linkage of the envelope proteinwith an antibody or a particular ligand for targeting to a receptor of aparticular cell-type. By inserting a sequence (including a regulatoryregion) of interest into the viral vector, along with another gene whichencodes the ligand for a receptor on a specific target cell, forexample, the vector is now target-specific.

The vector providing the viral env nucleic acid sequence is associatedoperably with regulatory sequences, e.g., a promoter or enhancer. Theregulatory sequence can be any eukaryotic promoter or enhancer,including for example, the Moloney murine leukemia viruspromoter-enhancer element, the human cytomegalovirus enhancer or thevaccinia P7.5 promoter. In some cases, such as the Moloney murineleukemia virus promoter-enhancer element, the promoter-enhancer elementsare located within or adjacent to the LTR sequences.

The heterologous or foreign nucleic acid sequence, such as the STAT-1αencoding polynucleotide sequence herein, is linked operably to aregulatory nucleic acid sequence. Preferably, the heterologous sequenceis linked to a promoter, resulting in a chimeric gene. The heterologousnucleic acid sequence may also be under control of either the viral LTRpromoter-enhancer signals or of an internal promoter, and retainedsignals within the retroviral LTR can still bring about efficientexpression of the transgene. Marker genes may be utilized to assay forthe presence of the vector, and thus, to confirm infection andintegration. The presence of a marker gene ensures the selection andgrowth of only those host cells which express the inserts. Typicalselection genes encode proteins that confer resistance to antibioticsand other toxic substances, e.g., histidinol, puromycin, hygromycin,neomycin, methotrexate, etc., and cell surface markers.

The vectors are introduced via transfection or infection into thepackaging cell line. The packaging cell line produces viral particlesthat contain the vector genome. Methods for transfection or infectionare well known by those of skill in the art. After cotransfection of thepackaging vectors and the transfer vector to the packaging cell line,the recombinant virus is recovered from the culture media and titered bystandard methods used by those of skill in the art. Thus, the packagingconstructs can be introduced into human cell lines by calcium phosphatetransfection, lipofection or electroporation, generally together with adominant selectable marker, such as neo, DHFR, Gln synthetase or ADA,followed by selection in the presence of the appropriate drug andisolation of clones. The selectable marker gene can be linked physicallyto the packaging genes in the construct.

Lentiviral transfer vectors Naldini et al. (1996), have been used toinfect human cells growth-arrested in vitro and to transduce neuronsafter direct injection into the brain of adult rats. The vector wasefficient at transferring marker genes in vivo into the neurons and longterm expression in the absence of detectable pathology was achieved.Animals analyzed ten months after a single injection of the vectorshowed no decrease in the average level of transgene expression and nosign of tissue pathology or immune reaction (Blomer et al., 1997). Thus,in the present invention, one may graft or transplant cells infectedwith the recombinant lentivirus ex vivo, or infect cells in vivo.

Other Viral Vectors. The development and utility of viral vectors forgene delivery is constantly improving and evolving. Other viral vectorssuch as poxvirus; e.g., vaccinia virus (Gnant et al., 1999; Gnant etal., 1999), alpha virus; e.g., sindbis virus, Semliki forest virus(Lundstrom, 1999), reovirus (Coffey et al., 1998) and influenza A virus(Neumann et al., 1999) are contemplated for use in the present inventionand may be selected according to the requisite properties of the targetsystem.

In certain embodiments, vaccinia viral vectors are contemplated for usein the present invention. Vaccinia virus is a particularly usefuleukaryotic viral vector system for expressing heterologous genes. Forexample, when recombinant vaccinia virus is properly engineered, theproteins are synthesized, processed and transported to the plasmamembrane. Vaccinia viruses as gene delivery vectors have recently beendemonstrated to transfer genes to human tumor cells, e.g., EMAP-II(Gnant et al., 1999), inner ear (Derby et al., 1999), glioma cells,e.g., p53 (Timiryasova et al., 1999) and various mammalian cells, e.g.,P-450 (U.S. Pat. No. 5,506,138). The preparation, growth andmanipulation of vaccinia viruses are described in U.S. Pat. No.5,849,304 and U.S. Pat. No. 5,506,138 (each specifically incorporatedherein by reference).

In other embodiments, sindbis viral vectors are contemplated for use ingene delivery. Sindbis virus is a species of the alphavirus genus(Garoff and Li, 1998) which includes such important pathogens asVenezuelan, Western and Eastern equine encephalitis viruses (Sawai etal., 1999; Mastrangelo et al., 1999). In vitro, sindbis virus infects avariety of avian, mammalian, reptilian, and amphibian cells. The genomeof sindbis virus consists of a single molecule of single-stranded RNA,11,703 nucleotides in length. The genomic RNA is infectious, is cappedat the 5′ terminus and polyadenylated at the 3′ terminus, and serves asmRNA. Translation of a vaccinia virus 26S mRNA produces a polyproteinthat is cleaved co- and post-translationally by a combination of viraland presumably host-encoded proteases to give the three virus structuralproteins, a capsid protein (C) and the two envelope glycoproteins (E1and PE2, precursors of the virion E2).

Three features of sindbis virus suggest that it would be a useful vectorfor the expression of heterologous genes. First, its wide host range,both in nature and in the laboratory. Second, gene expression occurs inthe cytoplasm of the host cell and is rapid and efficient. Third,temperature-sensitive mutations in RNA synthesis are available that maybe used to modulate the expression of heterologous coding sequences bysimply shifting cultures to the non-permissive temperature at varioustime after infection. The growth and maintenance of sindbis virus isknown in the art (U.S. Pat. No. 5,217,879, specifically incorporatedherein by reference).

Chimeric Viral Vectors. Chimeric or hybrid viral vectors are beingdeveloped for use in therapeutic gene delivery and are contemplated foruse in the present invention. Chimeric poxyiral/retroviral vectors(Holzer et al., 1999), adenoviral/retroviral vectors (Feng et al., 1997;Bilbao et al., 1999; Caplen et al., 1999) andadenoviral/adeno-associated viral vectors (Fisher et al., 1996; U.S.Pat. No. 5,871,982) have been described.

These “chimeric” viral gene transfer systems can exploit the favorablefeatures of two or more parent viral species. For example, Wilson etal., provide a chimeric vector construct which comprises a portion of anadenovirus, AAV 5′ and 3′ ITR sequences and a selected transgene,described below (U.S. Pat. No. 5,871,983, specifically incorporateherein by reference).

The adenovirus/AAV chimeric virus uses adenovirus nucleic acid sequencesas a shuttle to deliver a recombinant AAV/transgene genome to a targetcell. The adenovirus nucleic acid sequences employed in the hybridvector can range from a minimum sequence amount, which requires the useof a helper virus to produce the hybrid virus particle, to only selecteddeletions of adenovirus genes, which deleted gene products can besupplied in the hybrid viral production process by a selected packagingcell. At a minimum, the adenovirus nucleic acid sequences employed inthe pAdA shuttle vector are adenovirus genomic sequences from which allviral genes are deleted and which contain only those adenovirussequences required for packaging adenoviral genomic DNA into a preformedcapsid head. More specifically, the adenovirus sequences employed arethe cis-acting 5′ and 3′ inverted terminal repeat (ITR) sequences of anadenovirus (which function as origins of replication) and the native 5′packaging/enhancer domain, that contains sequences necessary forpackaging linear Ad genomes and enhancer elements for the E1 promoter.The adenovirus sequences may be modified to contain desired deletions,substitutions, or mutations, provided that the desired function is noteliminated.

The AAV sequences useful in the above chimeric vector are the viralsequences from which the rep and cap polypeptide encoding sequences aredeleted. More specifically, the AAV sequences employed are thecis-acting 5′ and 3′ inverted terminal repeat (ITR) sequences. Thesechimeras are characterized by high titer transgene delivery to a hostcell and the ability to stably integrate the transgene into the hostcell chromosome (U.S. Pat. No. 5,871,983, specifically incorporateherein by reference). In the hybrid vector construct, the AAV sequencesare flanked by the selected adenovirus sequences discussed above. The 5′and 3′ AAV ITR sequences themselves flank a selected transgene sequenceand associated regulatory elements, described below. Thus, the sequenceformed by the transgene and flanking 5′ and 3′ AAV sequences may beinserted at any deletion site in the adenovirus sequences of the vector.For example, the AAV sequences are desirably inserted at the site of thedeleted E1a/E1b genes of the adenovirus. Alternatively, the AAVsequences may be inserted at an E3 deletion, E2a deletion, and so on. Ifonly the adenovirus 5′ ITR/packaging sequences and 3′ ITR sequences areused in the hybrid virus, the AAV sequences are inserted between them.

The transgene sequence of the vector and recombinant virus can be agene, a nucleic acid sequence or reverse transcript thereof,heterologous to the adenovirus sequence, which encodes a protein,polypeptide or peptide fragment of interest. The transgene isoperatively linked to regulatory components in a manner which permitstransgene transcription. The composition of the transgene sequence willdepend upon the use to which the resulting hybrid vector will be put.For example, one type of transgene sequence includes a therapeutic genewhich expresses a desired gene product in a host cell. These therapeuticgenes or nucleic acid sequences typically encode products foradministration and expression in a patient in vivo or ex vivo to replaceor correct an inherited or non-inherited genetic defect or treat anepigenetic disorder or disease.

(x) Non-Viral Transformation

Suitable methods for nucleic acid delivery for transformation of anorganelle, a cell, a tissue or an organism for use with the currentinvention are believed to include virtually any method by which anucleic acid (e.g., DNA) can be introduced into an organelle, a cell, atissue or an organism, as described herein or as would be known to oneof ordinary skill in the art. Such methods include, but are not limitedto, direct delivery of DNA such as by injection (U.S. Pat. Nos.5,994,624, 5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932,5,656,610, 5,589,466 and 5,580,859, each incorporated herein byreference), including microinjection (Harland and Weintraub, 1985; U.S.Pat. No. 5,789,215, incorporated herein by reference); byelectroporation (U.S. Pat. No. 5,384,253, incorporated herein byreference); by calcium phosphate precipitation (Graham and Van Der Eb,1973; Chen and Okayama, 1987; Rippe et al., 1990); by using DEAE-dextranfollowed by polyethylene glycol (Gopal, 1985); by direct sonic loading(Fechheimer et al., 1987); by liposome mediated transfection (Nicolauand Sene, 1982; Fraley et al., 1979; Nicolau et al., 1987; Wong et al.,1980; Kaneda et al., 1989; Kato et al., 1991); by microprojectilebombardment (PCT Application Nos. WO 94/09699 and 95/06128; U.S. Pat.Nos. 5,610,042; 5,322,783, 5,563,055, 5,550,318, 5,538,877 and5,538,880, and each incorporated herein by reference); by agitation withsilicon carbide fibers (Kaeppler et al., 1990; U.S. Pat. Nos. 5,302,523and 5,464,765, each incorporated herein by reference); or byPEG-mediated transformation of protoplasts (Omirulleh et al., 1993; U.S.Pat. Nos. 4,684,611 and 4,952,500, each incorporated herein byreference); by desiccation/inhibition-mediated DNA uptake (Potrykus etal., 1985). Through the application of techniques such as these,organelle(s), cell(s), tissue(s) or organism(s) may be stably ortransiently transformed.

Injection. In certain embodiments, a nucleic acid may be delivered to anorganelle, a cell, a tissue or an organism via one or more injections(i.e., a needle injection), such as, for example, either subcutaneously,intradermally, intramuscularly, intervenously or intraperitoneally.Methods of injection of vaccines are well known to those of ordinaryskill in the art (e.g., injection of a composition comprising a salinesolution). Further embodiments of the present invention include theintroduction of a nucleic acid by direct microinjection. Directmicroinjection has been used to introduce nucleic acid constructs intoXenopus oocytes (Harland and Weintraub, 1985).

Electroporation. In certain embodiments of the present invention, anucleic acid is introduced into an organelle, a cell, a tissue or anorganism via electroporation. Electroporation involves the exposure of asuspension of cells and DNA to a high-voltage electric discharge. Insome variants of this method, certain cell wall-degrading enzymes, suchas pectin-degrading enzymes, are employed to render the target recipientcells more susceptible to transformation by electroporation thanuntreated cells (U.S. Pat. No. 5,384,253, incorporated herein byreference). Alternatively, recipient cells can be made more susceptibleto transformation by mechanical wounding.

Transfection of eukaryotic cells using electroporation has been quitesuccessful. Mouse pre-B lymphocytes have been transfected with humankappa-immunoglobulin genes (Potter et al., 1984), and rat hepatocyteshave been transfected with the chloramphenicol acetyltransferase gene(Tur-Kaspa et al., 1986) in this manner.

To effect transformation by electroporation in cells such as, forexample, plant cells, one may employ either friable tissues, such as asuspension culture of cells or embryogenic callus or alternatively onemay transform immature embryos or other organized tissue directly. Inthis technique, one would partially degrade the cell walls of the chosencells by exposing them to pectin-degrading enzymes (pectolyases) ormechanically wounding in a controlled manner. Examples of some specieswhich have been transformed by electroporation of intact cells includemaize (U.S. Pat. No. 5,384,253; Rhodes et al., 1995; D'Halluin et al.,1992), wheat (Zhou et al., 1993), tomato (Hou and Lin, 1996), soybean(Christou et al., 1987) and tobacco (Lee et al., 1989).

One also may employ protoplasts for electroporation transformation ofplant cells (Bates, 1994; Lazzeri, 1995). For example, the generation oftransgenic soybean plants by electroporation of cotyledon-derivedprotoplasts is described by Dhir and Widholm in International PatentApplication No. WO 9217598, incorporated herein by reference. Otherexamples of species for which protoplast transformation has beendescribed include barley (Lazerri, 1995), sorghum (Battraw et al.,1991), maize (Bhattacharjee et al., 1997), wheat (He et al., 1994) andtomato (Tsukada, 1989).

Calcium Phosphate. In other embodiments of the present invention, anucleic acid is introduced to the cells using calcium phosphateprecipitation. Human KB cells have been transfected with adenovirus 5DNA (Graham and Van Der Eb, 1973) using this technique. Also in thismanner, mouse L(A9), mouse C127, CHO, CV-1, BHK, NIH3T3 and HeLa cellswere transfected with a neomycin marker gene (Chen and Okayama, 1987),and rat hepatocytes were transfected with a variety of marker genes(Rippe et al., 1990).

DEAE-Dextran: In another embodiment, a nucleic acid is delivered into acell using DEAE-dextran followed by polyethylene glycol. In this manner,reporter plasmids were introduced into mouse myeloma and erythroleukemiacells (Gopal, 1985).

Sonication Loading. Additional embodiments of the present inventioninclude the introduction of a nucleic acid by direct sonic loading. LTK⁻fibroblasts have been transfected with the thymidine kinase gene bysonication loading (Fechheimer et al., 1987).

Liposome-Mediated Transfection. In a further embodiment of theinvention, a nucleic acid may be entrapped in a lipid complex such as,for example, a liposome. Liposomes are vesicular structurescharacterized by a phospholipid bilayer membrane and an inner aqueousmedium. Multilamellar liposomes have multiple lipid layers separated byaqueous medium. They form spontaneously when phospholipids are suspendedin an excess of aqueous solution. The lipid components undergoself-rearrangement before the formation of closed structures and entrapwater and dissolved solutes between the lipid bilayers (Ghosh andBachhawat, 1991). Also contemplated is an nucleic acid complexed withLipofectamine (Gibco BRL) or Superfect (Qiagen).

Liposome-mediated nucleic acid delivery and expression of foreign DNA invitro has been very successful (Nicolau and Sene, 1982; Fraley et al.,1979; Nicolau et al., 1987). The feasibility of liposome-mediateddelivery and expression of foreign DNA in cultured chick embryo, HeLaand hepatoma cells has also been demonstrated (Wong et al., 1980).

In certain embodiments of the invention, a liposome may be complexedwith a hemagglutinating virus (HVJ). This has been shown to facilitatefusion with the cell membrane and promote cell entry ofliposome-encapsulated DNA (Kaneda et al., 1989). In other embodiments, aliposome may be complexed or employed in conjunction with nuclearnon-histone chromosomal proteins (HMG-1) (Kato et al., 1991). In yetfurther embodiments, a liposome may be complexed or employed inconjunction with both HVJ and HMG-1. In other embodiments, a deliveryvehicle may comprise a ligand and a liposome.

Receptor-Mediated Transfection. Still further, a nucleic acid may bedelivered to a target cell via receptor-mediated delivery vehicles.These take advantage of the selective uptake of macromolecules byreceptor-mediated endocytosis that will be occurring in a target cell.In view of the cell type-specific distribution of various receptors,this delivery method adds another degree of specificity to the presentinvention.

Certain receptor-mediated gene targeting vehicles comprise a cellreceptor-specific ligand and a nucleic acid-binding agent. Otherscomprise a cell receptor-specific ligand to which the nucleic acid to bedelivered has been operatively attached. Several ligands have been usedfor receptor-mediated gene transfer (Wu and Wu, 1987; Wagner et al.,1990; Perales et al., 1994; Myers, EPO 0273085), which establishes theoperability of the technique. Specific delivery in the context ofanother mammalian cell type has been described (Wu and Wu, 1993;incorporated herein by reference). In certain aspects of the presentinvention, a ligand will be chosen to correspond to a receptorspecifically expressed on the target cell population.

In other embodiments, a nucleic acid delivery vehicle component of acell-specific nucleic acid targeting vehicle may comprise a specificbinding ligand in combination with a liposome. The nucleic acid(s) to bedelivered are housed within the liposome and the specific binding ligandis functionally incorporated into the liposome membrane. The liposomewill thus specifically bind to the receptor(s) of a target cell anddeliver the contents to a cell. Such systems have been shown to befunctional using systems in which, for example, epidermal growth factor(EGF) is used in the receptor-mediated delivery of a nucleic acid tocells that exhibit upregulation of the EGF receptor.

In still further embodiments, the nucleic acid delivery vehiclecomponent of a targeted delivery vehicle may be a liposome itself, whichwill preferably comprise one or more lipids or glycoproteins that directcell-specific binding. For example, lactosyl-ceramide, agalactose-terminal asialganglioside, have been incorporated intoliposomes and observed an increase in the uptake of the insulin gene byhepatocytes (Nicolau et al., 1987). It is contemplated that thetissue-specific transforming constructs of the present invention can bespecifically delivered into a target cell in a similar manner.

F. Expression Systems

Numerous prokaryote- and/or eukaryote-based systems can be employed foruse with the present invention to produce nucleic acid sequences, ortheir cognate polypeptides, proteins and peptides. Many such systems arecommercially and widely available.

The insect cell/baculovirus system can produce a high level of proteinexpression of a heterologous nucleic acid segment, such as described inU.S. Pat. Nos. 5,871,986 and 4,879,236, both herein incorporated byreference, and which can be bought, for example, under the name MAXBAC®2.0 from INVITROGEN® and BACPACK™ BACULOVIRUS EXPRESSION SYSTEM FROMCLONTECH®.

Other examples of expression systems include STRATAGENE®'S COMPLETECONTROL™ Inducible Mammalian Expression System, which involves asynthetic ecdysone-inducible receptor, or its pET Expression System, anE. coli expression system. Another example of an inducible expressionsystem is available from INVITROGEN®, which carries the T-REX™(tetracycline-regulated expression) System, an inducible mammalianexpression system that uses the full-length CMV promoter. INVITROGEN®also provides a yeast expression system called the Pichia methanolicaExpression System, which is designed for high-level production ofrecombinant proteins in the methylotrophic yeast Pichia methanolica. Oneof skill in the art would know how to express a vector, such as anexpression construct, to produce a nucleic acid sequence or its cognatepolypeptide, protein, or peptide.

Primary mammalian cell cultures may be prepared in various ways. Inorder for the cells to be kept viable while in vitro and in contact withthe expression construct, it is necessary to ensure that the cellsmaintain contact with the correct ratio of oxygen and carbon dioxide andnutrients but are protected from microbial contamination. Cell culturetechniques are well documented.

One embodiment of the foregoing involves the use of gene transfer toimmortalize cells for the production of proteins. The gene for theprotein of interest may be transferred as described above intoappropriate host cells followed by culture of cells under theappropriate conditions. The gene for virtually any polypeptide may beemployed in this manner. The generation of recombinant expressionvectors, and the elements included therein, are discussed above.Alternatively, the protein to be produced may be an endogenous proteinnormally synthesized by the cell in question.

Examples of useful mammalian host cell lines are Vero and HeLa cells andcell lines of Chinese hamster ovary, W138, BHK, COS-7, 293, HepG2,NIH3T3, RIN and MDCK cells. In addition, a host cell strain may bechosen that modulates the expression of the inserted sequences, ormodifies and process the gene product in the manner desired. Suchmodifications (e.g., glycosylation) and processing (e.g., cleavage) ofprotein products may be important for the function of the protein.Different host cells have characteristic and specific mechanisms for thepost-translational processing and modification of proteins. Appropriatecell lines or host systems can be chosen to insure the correctmodification and processing of the foreign protein expressed.

A number of selection systems may be used including, but not limited to,HSV thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase andadenine phosphoribosyltransferase genes, in tk−, hgprt− or aprt− cells,respectively. Also, anti-metabolite resistance can be used as the basisof selection for dhfr, that confers resistance to; gpt, that confersresistance to mycophenolic acid; neo, that confers resistance to theaminoglycoside G418; and hygro, that confers resistance to hygromycin.

G. Host Cells

As used herein, the terms “cell,” “cell line,” and “cell culture” may beused interchangeably. All of these terms also include their progeny,which is any and all subsequent generations. It is understood that allprogeny may not be identical due to deliberate or inadvertent mutations.In the context of expressing a heterologous nucleic acid sequence, “hostcell” refers to a prokaryotic or eukaryotic cell, and it includes anytransformable organisms that is capable of replicating a vector and/orexpressing a heterologous gene encoded by a vector. A host cell can, andhas been, used as a recipient for vectors. A host cell may be“transfected” or “transformed,” which refers to a process by whichexogenous nucleic acid is transferred or introduced into the host cell.A transformed cell includes the primary subject cell and its progeny.

Host cells may be derived from prokaryotes or eukaryotes, depending uponwhether the desired result is replication of the vector or expression ofpart or all of the vector-encoded nucleic acid sequences. Numerous celllines and cultures are available for use as a host cell, and they can beobtained through the American Type Culture Collection (ATCC), which isan organization that serves as an archive for living cultures andgenetic materials (www.atcc.org). An appropriate host can be determinedby one of skill in the art based on the vector backbone and the desiredresult. A plasmid or cosmid, for example, can be introduced into aprokaryote host cell for replication of many vectors. Bacterial cellsused as host cells for vector replication and/or expression includeDH5α, JM109, and KC8, as well as a number of commercially availablebacterial hosts such as SURE® Competent Cells and SOLOPACK™ Gold Cells(STRATAGENE®, La Jolla). Alternatively, bacterial cells such as E. coliLE392 could be used as host cells for phage viruses.

Examples of eukaryotic host cells for replication and/or expression of avector include HeLa, NIH3T3, Jurkat, 293, Cos, CHO, Saos, and PC12. Manyhost cells from various cell types and organisms are available and wouldbe known to one of skill in the art. Similarly, a viral vector may beused in conjunction with either a eukaryotic or prokaryotic host cell,particularly one that is permissive for replication or expression of thevector.

Some vectors may employ control sequences that allow it to be replicatedand/or expressed in both prokaryotic and eukaryotic cells. One of skillin the art would further understand the conditions under which toincubate all of the above described host cells to maintain them and topermit replication of a vector. Also understood and known are techniquesand conditions that would allow large-scale production of vectors, aswell as production of the nucleic acids encoded by vectors and theircognate polypeptides, proteins, or peptides.

H. Cell Propagation

Animal cells can be propagated in vitro in two modes: as non-anchoragedependent cells growing in suspension throughout the bulk of the cultureor as anchorage-dependent cells requiring attachment to a solidsubstrate for their propagation (i.e., a monolayer type of cell growth).Non-anchorage dependent or suspension cultures from continuousestablished cell lines are the most widely used means of large scaleproduction of cells and cell products. However, suspension culturedcells have limitations, such as tumorigenic potential and lower proteinproduction than adherent T-cells.

Large scale suspension culture of mammalian cells in stirred tanks is acommon method for production of recombinant proteins. Two suspensionculture reactor designs are in wide use—the stirred reactor and theairlift reactor. The stirred design has successfully been used on an8000 liter capacity for the production of interferon. Cells are grown ina stainless steel tank with a height-to-diameter ratio of 1:1 to 3:1.The culture is usually mixed with one or more agitators, based on bladeddisks or marine propeller patterns. Agitator systems offering less shearforces than blades have been described. Agitation may be driven eitherdirectly or indirectly by magnetically coupled drives. Indirect drivesreduce the risk of microbial contamination through seals on stirrershafts.

The airlift reactor, also initially described for microbial fermentationand later adapted for mammalian culture, relies on a gas stream to bothmix and oxygenate the culture. The gas stream enters a riser section ofthe reactor and drives circulation. Gas disengages at the culturesurface, causing denser liquid free of gas bubbles to travel downward inthe downcomer section of the reactor. The main advantage of this designis the simplicity and lack of need for mechanical mixing. Typically, theheight-to-diameter ratio is 10:1. The airlift reactor scales uprelatively easily, has good mass transfer of gases and generatesrelatively low shear forces.

The antibodies of the present invention are particularly useful for theisolation of antigens by immunoprecipitation. Immunoprecipitationinvolves the separation of the target antigen component from a complexmixture, and is used to discriminate or isolate minute amounts ofprotein. For the isolation of membrane proteins cells must besolubilized into detergent micelles. Non-ionic salts are preferred,since other agents such as bile salts, precipitate at acid pH or in thepresence of bivalent cations. Antibodies are and their uses arediscussed further, below.

III. Generating Antibodies Reactive with Notch3

In another aspect, the present invention contemplates an antibody thatis immunoreactive with a Notch3 molecule of the present invention, orany portion thereof. In particular, the invention contemplates usingpeptides having the sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3,SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8,optionally linked together or linked to a carrier molecule such askeyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Anantibody can be a polyclonal or a monoclonal antibody. In a preferredembodiment, an antibody is a monoclonal antibody. Means for preparingand characterizing antibodies are well known in the art (see, e.g.,Harlow and Lane, 1988).

Briefly, a polyclonal antibody is prepared by immunizing an animal withan immunogen comprising a polypeptide, of the present invention andcollecting antisera from that immunized animal. A wide range of animalspecies can be used for the production of antisera. Typically an animalused for production of anti-antisera is a non-human animal includingrabbits, mice, rats, hamsters, pigs or horses. Because of the relativelylarge blood volume of rabbits, a rabbit is a preferred choice forproduction of polyclonal antibodies.

Antibodies, both polyclonal and monoclonal, specific for isoforms ofantigen may be prepared using conventional immunization techniques, aswill be generally known to those of skill in the art. A compositioncontaining antigenic epitopes of the compounds of the present inventioncan be used to immunize one or more experimental animals, such as arabbit or mouse, which will then proceed to produce specific antibodiesagainst the compounds of the present invention. Polyclonal antisera maybe obtained, after allowing time for antibody generation, simply bybleeding the animal and preparing serum samples from the whole blood.

It is proposed that the monoclonal antibodies of the present inventionwill find useful application in standard immunochemical procedures, suchas ELISA and Western blot methods and in immunohistochemical proceduressuch as tissue staining, as well as in other procedures which mayutilize antibodies specific to Notch3-related antigen epitopes.Additionally, it is proposed that monoclonal antibodies specific to theparticular Notch3 of different species may be utilized in other usefulapplications

In general, both polyclonal and monoclonal antibodies against Notch3 maybe used in a variety of embodiments. For example, they may be employedin antibody cloning protocols to obtain cDNAs or genes encoding otherNotch3. They may also be used in inhibition studies to analyze theeffects of Notch3 related peptides in cells or animals. Anti-Notch3antibodies will also be useful in immunolocalization studies to analyzethe distribution of Notch3 during various cellular events, for example,to determine the cellular or tissue-specific distribution of Notch3polypeptides under different points in the cell cycle. A particularlyuseful application of such antibodies is in purifying native orrecombinant Notch3, for example, using an antibody affinity column. Theoperation of all such immunological techniques will be known to those ofskill in the art in light of the present disclosure.

Means for preparing and characterizing antibodies are well known in theart (see, e.g., Harlow and Lane, 1988; incorporated herein byreference). More specific examples of monoclonal antibody preparationare give in the examples below.

As is well known in the art, a given composition may vary in itsimmunogenicity. It is often necessary therefore to boost the host immunesystem, as may be achieved by coupling a peptide or polypeptideimmunogen to a carrier. Exemplary and preferred carriers are keyholelimpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albuminssuch as ovalbumin, mouse serum albumin or rabbit serum albumin can alsobe used as carriers. Means for conjugating a polypeptide to a carrierprotein are well known in the art and include glutaraldehyde,m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimide andbis-biazotized benzidine.

As also is well known in the art, the immunogenicity of a particularimmunogen composition can be enhanced by the use of non-specificstimulators of the immune response, known as adjuvants. Exemplary andpreferred adjuvants include complete Freund's adjuvant (a non-specificstimulator of the immune response containing killed Mycobacteriumtuberculosis), incomplete Freund's adjuvants and aluminum hydroxideadjuvant.

The amount of immunogen composition used in the production of polyclonalantibodies varies upon the nature of the immunogen as well as the animalused for immunization. A variety of routes can be used to administer theimmunogen (subcutaneous, intramuscular, intradermal, intravenous andintraperitoneal). The production of polyclonal antibodies may bemonitored by sampling blood of the immunized animal at various pointsfollowing immunization. A second, booster, injection may also be given.The process of boosting and titering is repeated until a suitable titeris achieved. When a desired level of immunogenicity is obtained, theimmunized animal can be bled and the serum isolated and stored, and/orthe animal can be used to generate mAbs.

MAbs may be readily prepared through use of well-known techniques, suchas those exemplified in U.S. Pat. No. 4,196,265, incorporated herein byreference. Typically, this technique involves immunizing a suitableanimal with a selected immunogen composition, e.g., a purified orpartially purified Notch3 protein, polypeptide or peptide or cellexpressing high levels of Notch3. The immunizing composition isadministered in a manner effective to stimulate antibody producingcells. Rodents such as mice and rats are preferred animals, however, theuse of rabbit, sheep frog cells is also possible. The use of rats mayprovide certain advantages (Goding, 1986), but mice are preferred, withthe BALB/c mouse being most preferred as this is most routinely used andgenerally gives a higher percentage of stable fusions.

Following immunization, somatic cells with the potential for producingantibodies, specifically B-lymphocytes (B-cells), are selected for usein the mAb generating protocol. These cells may be obtained frombiopsied spleens, tonsils or lymph nodes, or from a peripheral bloodsample. Spleen cells and peripheral blood cells are preferred, theformer because they are a rich source of antibody-producing cells thatare in the dividing plasmablast stage, and the latter because peripheralblood is easily accessible. Often, a panel of animals will have beenimmunized and the spleen of animal with the highest antibody titer willbe removed and the spleen lymphocytes obtained by homogenizing thespleen with a syringe. Typically, a spleen from an immunized mousecontains approximately 5×10⁷ to 2×10⁸ lymphocytes.

The antibody-producing B lymphocytes from the immunized animal are thenfused with cells of an immortal myeloma cell, generally one of the samespecies as the animal that was immunized. Myeloma cell lines suited foruse in hybridoma-producing fusion procedures preferably arenon-antibody-producing, have high fusion efficiency, and enzymedeficiencies that render then incapable of growing in certain selectivemedia which support the growth of only the desired fused cells(hybridomas).

Any one of a number of myeloma cells may be used, as are known to thoseof skill in the art (Goding, 1986; Campbell, 1984). For example, wherethe immunized animal is a mouse, one may use P3-X63/Ag8, P3-X63-Ag8.653,NS1/1.Ag 41, Sp210-Ag14, FO, NSO/U, MPC-11, MPC11-X45-GTG 1.7 andS194/5XX0 Bul; for rats, one may use R210.RCY3, Y3-Ag 1.2.3, IR983F and4B210; and U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6 are all usefulin connection with cell fusions.

Methods for generating hybrids of antibody-producing spleen or lymphnode cells and myeloma cells usually comprise mixing somatic cells withmyeloma cells in a 2:1 ratio, though the ratio may vary from about 20:1to about 1:1, respectively, in the presence of an agent or agents(chemical or electrical) that promote the fusion of cell membranes.Fusion methods using Sendai virus have been described (Kohler andMilstein, 1975; 1976), and those using polyethylene glycol (PEG), suchas 37% (v/v) PEG, by Gefter et al., (1977). The use of electricallyinduced fusion methods is also appropriate (Goding, 1986).

Fusion procedures usually produce viable hybrids at low frequencies,around 1×10⁻⁶ to 1×10⁻⁸. However, this does not pose a problem, as theviable, fused hybrids are differentiated from the parental, unfusedcells (particularly the unfused myeloma cells that would normallycontinue to divide indefinitely) by culturing in a selective medium. Theselective medium is generally one that contains an agent that blocks thede novo synthesis of nucleotides in the tissue culture media. Exemplaryand preferred agents are aminopterin, methotrexate, and azaserine.Aminopterin and methotrexate block de novo synthesis of both purines andpyrimidines, whereas azaserine blocks only purine synthesis. Whereaminopterin or methotrexate is used, the media is supplemented withhypoxanthine and thymidine as a source of nucleotides (HAT medium).Where azaserine is used, the media is supplemented with hypoxanthine.

The preferred selection medium is HAT. Only cells capable of operatingnucleotide salvage pathways are able to survive in HAT medium. Themyeloma cells are defective in key enzymes of the salvage pathway, e.g.,hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive.The B-cells can operate this pathway, but they have a limited life spanin culture and generally die within about two weeks. Therefore, the onlycells that can survive in the selective media are those hybrids formedfrom myeloma and B-cells.

This culturing provides a population of hybridomas from which specifichybridomas are selected. Typically, selection of hybridomas is performedby culturing the cells by single-clone dilution in microtiter plates,followed by testing the individual clonal supernatants (after about twoto three weeks) for the desired reactivity. The assay should besensitive, simple and rapid, such as radioimmunoassays, enzymeimmunoassays, cytotoxicity assays, plaque assays, dot immunobindingassays, and the like.

The selected hybridomas would then be serially diluted and cloned intoindividual antibody-producing cell lines, which clones can then bepropagated indefinitely to provide mAbs. The cell lines may be exploitedfor mAb production in two basic ways. A sample of the hybridoma can beinjected (often into the peritoneal cavity) into a histocompatibleanimal of the type that was used to provide the somatic and myelomacells for the original fusion. The injected animal develops tumorssecreting the specific monoclonal antibody produced by the fused cellhybrid. The body fluids of the animal, such as serum or ascites fluid,can then be tapped to provide mAbs in high concentration. The individualcell lines could also be cultured in vitro, where the mAbs are naturallysecreted into the culture medium from which they can be readily obtainedin high concentrations. mAbs produced by either means may be furtherpurified, if desired, using filtration, centrifugation and variouschromatographic methods such as HPLC or affinity chromatography.

IV. Methods of Therapy

The present invention also involves, in another embodiment, thetreatment of cancer. The types of cancer that may be treated, accordingto the present invention, is limited only by the involvement of Notch3.By involvement, it is not even a requirement that Notch3 be mutated orabnormal—the overexpression of this tumor suppressor may actuallyovercome other lesions within the cell. Thus, it is contemplated that awide variety of tumors may be treated using Notch3 therapy, includingcancers of the brain, lung, liver, spleen, kidney, lymph node, pancreas,small intestine, blood cells, colon, stomach, breast, endometrium,prostate, testicle, ovary, skin, head and neck, esophagus, bone marrow,blood or other tissue.

In many contexts, it is not necessary that the tumor cell be killed orinduced to undergo normal cell death or “apoptosis.” Rather, toaccomplish a meaningful treatment, all that is required is that thetumor growth be slowed to some degree. It may be that the tumor growthis completely blocked, however, or that some tumor regression isachieved. Clinical terminology such as “remission” and “reduction oftumor” burden also are contemplated given their normal usage.

A. Peptide Therapy

Another therapy approach is the provision, to a subject, of Notch3polypeptide, fragments, synthetic peptides, mimetics or other analogsthereof. The protein/peptide may be produced by recombinant expressionmeans or, if small enough, generated by an automated peptidesynthesizer. Formulations would be selected based on the route ofadministration and purpose including, but not limited to, liposomalformulations and classic pharmaceutical preparations.

B. Antibody Therapy

Applicants also contemplate the use of antibodies to Notch3, inparticular, to epitopes comprised in or represented by SEQ ID NO:1, SEQID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:7, or SEQ ID NO:8. Antibodies will be administered according tostandard protocols for passive immunotherapy. Administration protocolswould generally involve intratumoral, local or regional (to the tumor)administration, as well as systemic administration.

In addition, the antibody reagent may be altered, such that it will haveone or more improved properties. The antibody may be recombinant, i.e.,an antibody gene cloned into an expression cassette which is thenintroduced into a cell in which the antibody gene was not initiallycreated. The antibody may be single chain, a fragment (Fab, Fv, Vh,ScFv), chimeric or humanized.

C. Combined Therapies with Immunotherapy, Traditional Chemo- orRadiotherapy

Tumor cell resistance to DNA damaging agents represents a major problemin clinical oncology. One goal of current cancer research is to findways to improve the efficacy of chemo- and radiotherapy. One way is bycombining such traditional therapies with gene therapy. For example, theherpes simplex-thymidine kinase (HS-tk) gene, when delivered to braintumors by a retroviral vector system, successfully inducedsusceptibility to the antiviral agent ganciclovir (Culver et al., 1992).In the context of the present invention, it is contemplated that Notch3peptide or antibody therapy could be used similarly in conjunction withchemo- or radiotherapeutic intervention. It also may prove effective tocombine a Notch3-directed therapy with another cancer therapy.

To kill cells, inhibit cell growth, inhibit metastasis, inhibitangiogenesis or otherwise reverse or reduce the malignant phenotype oftumor cells, using the methods and compositions of the presentinvention, one would generally contact a “target” cell with a Notch3peptide or antibody and at least one other agent. These compositionswould be provided in a combined amount effective to kill or inhibitproliferation of the cell. This process may involve contacting the cellswith a Notch3 peptide or antibody and the agent(s) or factor(s) at thesame time. This may be achieved by contacting the cell with a singlecomposition or pharmacological formulation that includes both agents, orby contacting the cell with two distinct compositions or formulations,at the same time, wherein one composition includes the expressionconstruct and the other includes the agent.

Alternatively, the Notch3 peptide or antibody therapy treatment mayprecede or follow the other agent treatment by intervals ranging fromminutes to weeks. In embodiments where the other agent and expressionconstruct are applied separately to the cell, one would generally ensurethat a significant period of time did not expire between the time ofeach delivery, such that the agent and a Notch3 peptide or antibodywould still be able to exert an advantageously combined effect on thecell. In such instances, it is contemplated that one would contact thecell with both modalities within about 12-24 hours of each other and,more preferably, within about 6-12 hours of each other, with a delaytime of only about 12 hours being most preferred. In some situations, itmay be desirable to extend the time period for treatment significantly,however, where several days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2,3, 4, 5, 6, 7 or 8) lapse between the respective administrations.

It also is conceivable that more than one administration of eitherNotch3 peptide or antibody or the other agent will be desired. Variouscombinations may be employed, where Notch3 (peptide or antibody) is “A”and the other agent is “B”, as exemplified below:

-   -   A/B/A B/A/B B/B/A A/A/B B/A/A A/B/B B/B/B/A B/B/A/B    -   A/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B B/B/B/A    -   A/A/A/B B/A/A/A A/B/A/A A/A/B/A A/B/B/B B/A/B/B B/B/A/B        Other combinations are contemplated. Again, to achieve cell        killing, both agents are delivered to a cell in a combined        amount effective to kill the cell.

Agents or factors suitable for use in a combined therapy are anychemical compound or treatment method that induces DNA damage whenapplied to a cell. Such agents and factors include radiation and wavesthat induce DNA damage such as, γ-irradiation, X-rays, UV-irradiation,microwaves, electronic emissions, and the like. A variety of chemicalcompounds, also described as “chemotherapeutic agents,” function toinduce DNA damage, all of which are intended to be of use in thecombined treatment methods disclosed herein. Chemotherapeutic agentscontemplated to be of use, include, e.g., adriamycin, 5-fluorouracil(5FU), etoposide (VP-16), camptothecin, actinomycin-D, mitomycin C,cisplatin (CDDP) and even hydrogen peroxide. The invention alsoencompasses the use of a combination of one or more DNA damaging agents,whether radiation-based or actual compounds, such as the use of X-rayswith cisplatin or the use of cisplatin with etoposide. In certainembodiments, the use of cisplatin in combination with a Notch3expression construct is particularly preferred as this compound.

In treating cancer according to the invention, one would contact thetumor cells with an agent in addition to the expression construct. Thismay be achieved by irradiating the localized tumor site with radiationsuch as X-rays, UV-light, γ-rays or even microwaves. Alternatively, thetumor cells may be contacted with the agent by administering to thesubject a therapeutically effective amount of a pharmaceuticalcomposition comprising a compound such as, adriamycin, 5-fluorouracil,etoposide, camptothecin, actinomycin-D, mitomycin C, or more preferably,cisplatin. The agent may be prepared and used as a combined therapeuticcomposition, or kit, by combining it with a Notch3 expression construct,as described above.

Agents that directly cross-link nucleic acids, specifically DNA, areenvisaged to facilitate DNA damage leading to a synergistic,antineoplastic combination with Notch3. Agents such as cisplatin, andother DNA alkylating agents may be used. Cisplatin has been widely usedto treat cancer, with efficacious doses used in clinical applications of20 mg/m² for 5 days every three weeks for a total of three courses.Cisplatin is not absorbed orally and must therefore be delivered viainjection intravenously, subcutaneously, intratumorally orintraperitoneally.

Agents that damage DNA also include compounds that interfere with DNAreplication, mitosis and chromosomal segregation. Such chemotherapeuticcompounds include adriamycin, also known as doxorubicin, etoposide,verapamil, podophyllotoxin, and the like. Widely used in a clinicalsetting for the treatment of neoplasms, these compounds are administeredthrough bolus injections intravenously at doses ranging from 25-75 mg/m²at 21 day intervals for adriamycin, to 35-50 mg/m² for etoposideintravenously or double the intravenous dose orally.

Agents that disrupt the synthesis and fidelity of nucleic acidprecursors and subunits also lead to DNA damage. As such a number ofnucleic acid precursors have been developed. Particularly useful areagents that have undergone extensive testing and are readily available.As such, agents such as 5-fluorouracil (5-FU), are preferentially usedby neoplastic tissue, making this agent particularly useful fortargeting to neoplastic cells. Although quite toxic, 5-FU, is applicablein a wide range of carriers, including topical, however intravenousadministration with doses ranging from 3 to 15 mg/kg/day being commonlyused.

Other factors that cause DNA damage and have been used extensivelyinclude what are commonly known as γ-rays, X-rays, and/or the directeddelivery of radioisotopes to tumor cells. Other forms of DNA damagingfactors are also contemplated such as microwaves and UV-irradiation. Itis most likely that all of these factors effect a broad range of damageDNA, on the precursors of DNA, the replication and repair of DNA, andthe assembly and maintenance of chromosomes. Dosage ranges for X-raysrange from daily doses of 50 to 200 roentgens for prolonged periods oftime (3 to 4 weeks), to single doses of 2000 to 6000 roentgens. Dosageranges for radioisotopes vary widely, and depend on the half-life of theisotope, the strength and type of radiation emitted, and the uptake bythe neoplastic cells.

The skilled artisan is directed to “Remington's Pharmaceutical Sciences”15th Edition, chapter 33, in particular pages 624-652. Some variation indosage will necessarily occur depending on the condition of the subjectbeing treated. The person responsible for administration will, in anyevent, determine the appropriate dose for the individual subject.Moreover, for human administration, preparations should meet sterility,pyrogenicity, general safety and purity standards as required by FDAOffice of Biologics standards.

The inventors propose that the local or regional delivery of Notch3expression constructs to patients with cancer will be a very efficientmethod for treating the clinical disease. Similarly, the chemo- orradiotherapy may be directed to a particular, affected region of thesubjects body. Alternatively, systemic delivery of expression constructand/or the agent may be appropriate in certain circumstances, forexample, where extensive metastasis has occurred.

In addition to combining Notch3 therapies with chemo- andradiotherapies, it also is contemplated that combination with other genetherapies will be advantageous. For example, targeting of Notch3 and p53mutations at the same time may produce an improved anti-cancertreatment. Any other tumor-related gene conceivably can be targeted inthis manner, for example, p21, Rb, APC, DCC, NF-1, NF-2, BCRA2, p16,FHIT, WT-1, MEN-I, MEN-II, BRCA1, VHL, FCC, MCC, ras, myc, neu, raf erb,src, fms, jun, trk, ret, gsp, hst, bcl and abl.

It also should be pointed out that any of the foregoing therapies mayprove useful by themselves in treating a Notch3. In this regard,reference to chemotherapeutics and non-Notch3 gene therapy incombination should also be read as a contemplation that these approachesmay be employed separately.

D. Formulations and Routes for Administration to Patients

Where clinical applications are contemplated, it will be necessary toprepare pharmaceutical compositions—a Notch3 peptide, or antibody—in aform appropriate for the intended application. Generally, this willentail preparing compositions that are essentially free of pyrogens, aswell as other impurities that could be harmful to humans or animals.

One will generally desire to employ appropriate salts and buffers torender delivery compositions stable and allow for uptake by targetcells. Aqueous compositions of the present invention comprise aneffective amount of the Notch3 peptide or antibody to cells/a subject,dissolved or dispersed in a pharmaceutically acceptable carrier oraqueous medium. Such compositions also are referred to as inocula. Thephrase “pharmaceutically or pharmacologically acceptable” refer tomolecular entities and compositions that do not produce adverse,allergic, or other untoward reactions when administered to an animal ora human. As used herein, “pharmaceutically acceptable carrier” includesany and all solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents and the like.The use of such media and agents for pharmaceutically active substancesis well know in the art. Except insofar as any conventional media oragent is incompatible with the vectors or cells of the presentinvention, its use in therapeutic compositions is contemplated.Supplementary active ingredients also can be incorporated into thecompositions.

The active compositions of the present invention may include classicpharmaceutical preparations. Administration of these compositionsaccording to the present invention will be via any common route so longas the target tissue is available via that route. This includes oral,nasal, buccal, rectal, vaginal or topical. Alternatively, administrationmay be by orthotopic, intradermal, subcutaneous, intramuscular,intraperitoneal or intravenous injection. Such compositions wouldnormally be administered as pharmaceutically acceptable compositions,described supra. Of particular interest is direct intratumoraladministration, perfusion of a tumor, or administration local orregional to a tumor, for example, in the local or regional vasculatureor lymphatic system, or in a resected tumor bed.

The active compounds may also be administered parenterally orintraperitoneally. Solutions of the active compounds as free base orpharmacologically acceptable salts can be prepared in water suitablymixed with a surfactant, such as hydroxypropylcellulose. Dispersions canalso be prepared in glycerol, liquid polyethylene glycols, and mixturesthereof and in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), suitable mixtures thereof,and vegetable oils. The proper fluidity can be maintained, for example,by the use of a coating, such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial an antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutical active substances is well knownin the art. Except insofar as any conventional media or agent isincompatible with the active ingredient, its use in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions.

For oral administration the Notch3 peptides or antibodies of the presentinvention may be incorporated with excipients and used in the form ofnon-ingestible mouthwashes and dentifrices. A mouthwash may be preparedincorporating the active ingredient in the required amount in anappropriate solvent, such as a sodium borate solution (Dobell'sSolution). Alternatively, the active ingredient may be incorporated intoan antiseptic wash containing sodium borate, glycerin and potassiumbicarbonate. The active ingredient may also be dispersed in dentifrices,including: gels, pastes, powders and slurries. The active ingredient maybe added in a therapeutically effective amount to a paste dentifricethat may include water, binders, abrasives, flavoring agents, foamingagents, and humectants.

The compositions of the present invention may be formulated in a neutralor salt form. Pharmaceutically-acceptable salts include the acidaddition salts (formed with the free amino groups of the protein) andwhich are formed with inorganic acids such as, for example, hydrochloricor phosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups canalso be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, histidine, procaine and thelike.

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeuticallyeffective. The formulations are easily administered in a variety ofdosage forms such as injectable solutions, drug release capsules and thelike. For parenteral administration in an aqueous solution, for example,the solution should be suitably buffered if necessary and the liquiddiluent first rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, sterile aqueous media which can be employed will be known tothose of skill in the art in light of the present disclosure. Forexample, one dosage could be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion, (see for example, “Remington's PharmaceuticalSciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variationin dosage will necessarily occur depending on the condition of thesubject being treated. The person responsible for administration will,in any event, determine the appropriate dose for the individual subject.Moreover, for human administration, preparations should meet sterility,pyrogenicity, general safety and purity standards as required by FDAOffice of Biologics standards.

V. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1

Notch3 Is Expressed in Resected Human Lung Cancers. The clinicalrelevance of a pathway to tumorigenesis often depends on the prevalenceof its dysregulation. To begin to assess the prevalence of Notch3involvement in lung cancer, the inventors investigated the frequency ofNotch3 overexpression in resected lung cancer. They usedimmunohistochemistry (IHC), with a characterized and validated Notch3antibody recognizing the extracellular domain (Joutel et al., 2000), inresected lung tumor tissues. For this experiment, the inventors usedtumor tissue arrays produced as part of the Vanderbilt SPORE initiative.When the Notch3 antibody targeting the extracellular domain was used, apattern of cytoplasmic and membranous staining was observed in therepresentative adenocarcinoma and squamous cell carcincoma (FIG. 3,panel A and C). No staining was observed in a neuroendocrine tumor andnormal lung tissue (panel B and D), suggesting that Notch3 dysregulationis specific to cancer. The staining was scored on a composite scale of 0to 4 by two independent investigators, including one pathologist. Nostaining was scored 0, whereas slight positivity equivalent tobackground stain was scored as 1. Tumors that scored 2 or higher wereconsidered positive. Fractional positivity was nearly 100% in manytumors with high Notch3 expression. Of the 207 resected tumors, 39% hadhigh Notch3 expression (Table 7). Each tumor was represented intriplicate, and the final score per tumor was calculated by averagingthe score of three samples.

TABLE 7 Expression of Notch3 and EGFr By IHC Using Microtissue ArraysNotch3+ EGFr+ N n % n % Adenocarcinomas 87 32 37% 69 79% NeuroendocrineCarcinoid 10 2 20% 5 50% Large Cell 7 1 14% 2 29% Small Cell 4 1 25% 375% Squamous Cell Carcinomas 88 40 45% 81 92% Large Cell 11 4 36% 8 73%207 80 39% 168 81%

The frequency of Notch3 overexpression in lung tumors is higher than thefrequency of HER2/neu (16%) and k-Ras mutations (16%), and comparable toEGFr (13-80%) (Hirsch et al., 2002; Rodenhuis et al., 1997; Meert etal., 2002). No information is available with regard to the frequency ofoverexpression of other members of the Notch family or their receptorsin human lung carcinomas. The lack of high quality antibodies to theNotch receptors, Jagged1, and Delta-like-1, -3 and -4 makes it difficultto determine their frequency in fresh tumor tissues. In both C. elegansand Drosophila, the Notch pathway crosstalks with the EGF pathway incell fate determination during development. The inventors thus examinedthe correlation between Notch3 and EGFr expression. The frequency ofEGFr expression in our tumor tissue arrays was 81%. 19% of EGFr positivetumors are Notch3 negative, whereas 43% of EGFr positive tumors are alsoNotch3 positive (p<0.0001 using Pearson correlation coefficient) (Harukiet al., 2005). This highly statistical association suggests that theNotch and EGF pathways are cooperative in lung tumorigenesis.

Ectopic Notch3 Expression Inhibits Terminal Differentiation inDeveloping Lungs of Transgenic Mice. To evaluate the potentialtransforming activity of Notch3 in vivo, the inventors studied theeffect of activated Notch3 using a lung-specific, human SP-C promoter(Glasser et al., 1991; Lardelli et al., 1996). Theconstitutively-activate Notch3 allowed the inventors to assess theeffect of ectopic expression of Notch3 without depending on ligandexpression in the epithelium, since Jagged1 expression becomesrestricted to endothelium as the lung matures (Taichman et al., 2002).Furthermore, constitutive expression of Notch3 also better mimics themany dysregulated pathways observed in cancer. The inventors observedperinatal lethality, and thus no surviving animals expresses the N3ICtransgene.

Because of the crucial role of the Notch family in development,expression of a constitutively active Notch3 transgene could potentiallydisturb normal lung development. Therefore, the transgenic mice weresacrificed prior to birth, at E18.5. Of the 182 embryos at E18.5gestation collected from 32 pregnant mothers, 10 were transgenic, asdetermined by PCR and Southern blot analysis. The inventors observedaltered lung morphogenesis, altered terminal sac morphology, andabnormally abundant mesenchyme, and no type I pneumocytes when comparedwith control mice (FIGS. 4A-D). Despite the important role of the Notchsignaling pathway in vascular development, no alteration invasculogenesis was observed in the transgenics using the PECAM-1antibody (data not shown), suggesting that the mesenchymaldifferentiation was influenced by the abnormally developing epithelium,and not by alterations of angiogenesis. While the majority of cuboidalcells lining the peripheral airways are pneumocytes based on their TTF1positivity, they failed to demonstrate markers of mature type IIpneumocytes, such as surfactant proteins C and B, markers for Claracells (Clara cells secretory protein), or neuroendocrine cells(calcitonin gene-related peptide) suggesting that they were immaturetype II cells (data not shown).

Thus, this transgenic model provides evidence that dysregulation ofNotch3 signaling in the developing lung affects morphogenesis andterminal differentiation of the lung epithelium. Prenatal activation ofNotch3 in the peripheral epithelium of the lung in our SP-C-N3IC mousemodel leads to nonviable newborn pups, making it impossible to evaluatetumor progression. An inducible transgenic model that allows activationof Notch3 signaling in the postnatal period will be more effectivelyrecapitulate the somatic activation of potential oncogenes observed inhuman lung cancer. This approach is part of the proposal in our R01funding. Regardless, the ectopic expression of Notch3 appears to inhibitterminal differentiation of type I pneumocytes and results in metaplasiaof the immature respiratory epithelium, supporting a potential role ofNotch3 in lung cancer formation.

Notch3 Inhibition Inhibits the Tumor Phenotype. To test their hypothesisthat Notch3 plays an important role in the pathogenesis of lung cancer,the inventors used the approach of uncoupling its ligand binding andsignaling functions (Rebay et al., 1993). To inhibit Notch3 activation,the inventors created a dominant-negative (DN) construct. Onecharacteristic feature of malignant transformation is the ability oftumor cells to proliferate in the absence of adhesion, as measured bythe soft-agar assay for colony formation. The inventors demonstratedthat inhibition of Notch3 signaling by the DN construct dramaticallydecreases the ability of HCC2429 and H460 to form colonies in soft agar(FIG. 5A). Furthermore, colonies formed by the DN clones are markedlysmaller than those seen with the vector control (VC). Transformed cellsoften have a relaxed serum or growth factor requirement forproliferation in monolayer culture (Holley, 1975). In serum-free medium,the DN clones failed to proliferate when compared to the vector controls(FIG. 5B). In summary, these observations demonstrated that inhibitingthe Notch pathway using DN constructs reduces the tumor phenotype,supporting an oncogenic role for Notch3 in lung cancer.

Since Jagged1 is known to bind to other Notch receptors, it is possiblethat the antitumor effect observed when DN receptor is notNotch3-specific, since its mechanism of action is to sequester ligand(Shimizu et al., 2000). To determine whether specific inhibition ofNotch3 activation can inhibit tumor phenotype, the inventors used siRNAto specifically knock down Notch3 expression. The inventors show thatinhibiting Notch3 results in the lack of focus formation, furthersupporting the role of Notch3 in lung cancer pathogenesis (FIG. 6).

A γ-Secretase Inhibitor Reduces Proliferation in Lung Cancer Cells.Proteolytic processing of Notch receptors is required for activation. Aspreviously described, three proteolytic cleavage sites are involved inenabling Notch signaling. In the final step, the membrane-associatedNotch fragment is cleaved within its transmembrane domain by aγ-secretase-containing protein complex. Mammalian presenilins (PS-1 andPS-2) are polytopic transmembrane proteins that appear to function asaspartyl proteases within a multiprotein, γ-secretase complex (Wolfe andKopan, 2004). Presenilins provide the active site of the proteolyticactivity. This last cleavage releases the Notch intracellular domain,initiating CBF-1-mediated signaling. Thus, pharmacologic interventionthat inhibits the activity of the γ-secretase proteases can potentiallyinhibit tumor growth in Notch-dependent cancer. Interestingly, at thesame time that presenilins were shown to be essential for Notchsignaling, they were discovered as susceptibility loci for Alzheimer'sdisease (Levitan and Greenwald, 1995). The pathogenesis of Alzheimer'sdisease is believed to be the accumulation of amyloid β-peptide (Aβ),which is derived from proteolytic processing of the β-amyloid precursorprotein (APP) by β- and γ-secretases. Since inhibition of the Notch3pathway using the DN construct resulted in the loss of the tumorphenotype, the inventors wanted to examine the effect of γ-secretaseinhibitors on tumor growth. The inventors treated HCC2429 cells with GSI(Gamma Secretase Inhibitor), a commercially available γ-secretaseinhibitor (Calbiochem). The inventors observed that GSI inhibits tumorproliferation with an IC₅₀ of about 1 μM in comparison to DMSO (FIG.7A). However, in the presence of low serum the inhibition increasesalmost 1 log. This finding is similar to what was observed when the DNconstruct was used in HCC2429. This observation suggests that theantiproliferative activity observed is Notch-dependent. The inventorsalso observed inhibition of proliferation in other lung cancer celllines expressing Notch3 (data not shown). Biochemically, treatment withthese inhibitors also resulted in the loss of activated Notch3, asmeasured by the levels of the N3ICD (FIG. 7B). Stable expression ofNotch3 siRNA also leads to loss of sensitivity to γ-secretase inhibition(FIGS. 7C, 7D).

To assess the effect of pharmacologically inhibiting Notch activation invivo, the inventors treated subcutaneous xenografts with MRK003, aγ-secretase inhibitor from Merck, Inc. & Co. Based on the manufacturer'srecommended dose, the mice were treated with 100 mg/kg orally for 3consecutives days per week once the tumors were palpable. At the end ofa 2-week treatment, the animals were then euthanized, and the tumorswere harvested. A reduction of tumor size was seen in animals treatedwith the inhibitor, with a concomitant reduction in activated Notch3(N3ICD) (FIGS. 8A-D).

Notch3 Inhibition Induces Apoptosis. One hallmark of cancer isresistance to apoptosis. Genetic and pharmacologic inhibition of theNotch pathway in other tumors has been shown to induce apoptosis (Qin etal., 2004; Curry et al., 2005; Shelly et al., 1999). The inventorsexamined whether inhibiting the Notch3 pathway can induce apoptosis. Theinventors noted that in the HCC2429 clone expressing thedominant-negative receptor, the inhibition of the Notch3 pathway appearsto render the tumor cells sensitive to apoptosis in the presence ofserum starvation compared to vector control (FIG. 9A). One mechanism ofapoptosis induction is the down-regulation of Akt phosphorylation. FIG.9B shows that the level of phosphorylated Akt does indeed decrease whenthe DN Notch3 construct is used. The inventors also demonstrated thatinhibition of Notch3 using siRNA results in the down-regulation ofBcl-xL (24 and 48 hours) and enhances the upregulation of cleaved PARP(48 hours), suggesting that Notch3 plays and important role in tumorsurvival (FIG. 9C).

Notch3 Crosstalks with the MAPK Pathway. In mammals, Notch receptorssignal primarily by binding to CBF-1 and related transcriptionco-activators. However, as mentioned previously, the Notch and EGF/MAPKpathways are known to interact in developing vertebrates andinvertebrates. Thus, since the MAPK pathway, and in particular the ERKsubfamily, also plays a prominent role in cellular response to growthfactors, and is often altered in cancer, The inventors examined whetherNotch3 alters ERK signaling in lung cancer cells. Using the DN receptor,they showed that inhibiting Notch3 downregulates MAPK, and conversely,that MAPK is upregulated when the cells were transfected with activatedNotch3 intracellular domain (FIGS. 10A, 10B). Moreover, lower levels ofactivated MAPK (p44/p42) expression in the unstimulated DN clones and amarkedly higher level of activation with growth factor induction in theVC in comparison to the DN clone suggest that the disruption of theNotch3 signaling pathway renders lung cancer cells more resistant togrowth-factor-dependent MAPK activation. This observation has beenconfirmed in two other lung cancer cell lines, H460 and H1819,transfected with the DN construct.

Interestingly, with prolonged exposure to serum (60 minutes), theHCC2429 clone expressing the dominant-negative construct shows thatp44/p42 phosphorylation was attenuated significantly compared to vectorcontrol (FIG. 10A). Prolonged exposure to growth factors and activationof the p44/p42 cascade can induce the expression of MAPK phosphatases-1and -2 (MKP-1/-2) as part of a negative feedback loop and result in thedown-regulation of the MAPK pathway (Traverse et al., 1992; Plows etal., 2002; Haneda et al., 1999; Brondello et al., 1997). Down-regulationof MKPs or resistance to dephosphorylation by MKPs are often observed inhuman tumors, and Notch3 may have a role in suppressing MKP expression(Sivaraman et al., 1997; Magi-galluzzi et al., 1997; Barry et al.,2001). Recent data demonstrate that one mechanism by which Notchantagonizes EGF in developing C. elegans is the upregulation of LIP-1, ahomolog of mammalian MAPK phosphatases (Berset et al., 2001). SinceDrosophila and C. elegans Notch have the highest homology to humanNotch1, and Notch3 appears to antagonize Notch1, it follows that Notch3might suppress MAPK phosphatase expression in cancer cells. To test thishypothesis, the inventors quantitated the level of MKP-1 using real-timePCR following serum induction in HCC2429 stably transfected with DN andVC. Higher levels of MKP-1 were observed in the DN clones (FIG. 10C).This finding suggests that Notch3 also modulates MAPK activation throughMKP-1 transcriptional control.

EGFr Inhibitors Enhance the Anti-proliferation Effect of Notch3Inhibition. The inventors previously showed that Notch3 expressionpositively correlates with EGFR expression in our tumor tissue array.The also showed that Notch3 cooperates with the MAPK pathway. Based onexisting literature and their observations, the inventors hypothesizedthat Notch3 acts synergistically with the EGFR pathway in the promotionand the survival of lung cancer cells and that combining inhibitors ofboth pathways will have synergistic therapeutic value. To test thishypothesis, the inventors examined whether inhibiting the Notch3 pathwayincreases tumor inhibition when lung cancer cells are treated with anEGFR tyrosine kinase inhibitor, AG1478. When HCC2429 cells weremaintained in EGF-supplemented media and treated with increasing dosesof AG1478, the clones transfected with the DN construct showed a 40-foldincrease in sensitivity to EGFR inhibitors, that is, the IC₅₀ wasreduced from 8.3 μM to 0.2 μM (FIG. 11A). In H460, a lung cancer cellline that has lower Notch3 expression as well as a k-Ras mutation (datanot shown) and is more resistant to AG1478 (IC₅₀ of 23.8 μM), theinhibition of the Notch3 pathway also reduces cancer cell survival byapproximately two-fold (IC₅₀ of 12.1 μM, FIG. 10A). These data provideevidence that Notch3 activation may decrease a tumor's dependence on theEGF pathway and thus further decrease the sensitivity to EGFR tyrosinekinase inhibitors. Synergism can also be observed when the γ-secretaseinhibitor L-685,458 is added to AG1478 (FIG. 11B). Using the soft-agarcolony assay, the inventors also demonstrate additive effects bycombining MRK003, another γ-secretase inhibitor, with an EGFR inhibitor(FIGS. 12A, 12B). From a therapeutic standpoint, Notch3 is a good targetfor therapeutic intervention both alone and in combination with growthfactor receptor inhibitors. Since about 80% of lung carcinomas expressEGFR, but far fewer respond to kinase inhibition, our data suggest thatadding a Notch3 inhibitor will improve the response rate in patientstreated with EGFR inhibitors.

Notch3 peptides induce apoptosis, inhibit Notch3-regulated gene Hey1,and interrupt signaling through binding to ligand Jagged1. FIG. 13 is arepresentative experiment showing that the peptides induce apoptosis byAnnexin V staining through screening using an FMAT system. Each of 155different peptides were assayed in quadruplicate, and only thosepeptides that produced a significant increase of fluorescence signal inall 4 wells were considered potentially positive or capable of inducingapoptosis. The bar graphs here reflect fluorescence counts. FIG. 14Ashows HCC2429 treated with Notch3 peptides N16, N17, N102, N103, N132,with induction of apoptosis by peptides compared to control. Treatmentwith peptides also reduced transcription of Notch3-dependent gene Hey1as determined by real-time RT-PCR (FIG. 14B). Of note, N17 peptide bothdemonstrates highest apoptotic activity and best reduction in Hey1transcription. FIG. 15 shows HEK cells transfected with Jagged1-HA andtreated with Notch3 peptides. The peptides were then immunoprecipitatedfrom cell lysate with streptavidin beads and immunoblotted with anti-HAantibody. This suggests that peptide induces apoptosis via binding toligand Jagged1 and preventing activation of Notch3 receptor.

Sera from mice immunized with Notch3 recombindant protein inhibit Notch3activation. FIG. 16 shows an immunoblot that demonstrates that sera frommice #2, 3, 4, 5, 6 can reduced cleavage of Notch3 ICD (Tx) in Notch3expressing cell line HCC2429 as compared a control (C). Recombinantprotein representing EGF-like repeats 21-22 and encompassing sequenceCTNLAGSFSCTCHGGYTGPSCDQDINDCDPNPCLNGGS was used to immunize AJ andBALB/c mice.

Recombinant Fc-fusion Notch3 proteins inhibit Notch3 activation andinduces apoptosis in vitro. FIG. 17A shows Fc-fusion protein comprisedfor N16-17 and N132 sequences inhibits Notch3 activation, while FIG. 17Bshows that purified recombinant N16-17-Fc protein induces apoptosis ascompared to control and Fc control after 40 hrs treatment. This studyfurther supports the hypothesis that these regions of Notch3 areimportant for ligand interaction, and that disruption of thisinteraction using decoy recombinant receptor can inhibit Notch3activation.

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and/or methods and in the steps or in the sequence of stepsof the method described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents that are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

IX. REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference:

-   U.S. Pat. No. 4,196,265-   U.S. Pat. No. 4,554,101-   U.S. Pat. No. 4,683,202-   U.S. Pat. No. 4,684,611-   U.S. Pat. No. 4,879,236-   U.S. Pat. No. 4,952,500-   U.S. Pat. No. 5,217,879-   U.S. Pat. No. 5,302,523-   U.S. Pat. No. 5,322,783-   U.S. Pat. No. 5,384,253-   U.S. Pat. No. 5,464,765-   U.S. Pat. No. 5,506,138-   U.S. Pat. No. 5,538,877-   U.S. Pat. No. 5,538,880-   U.S. Pat. No. 5,550,318-   U.S. Pat. No. 5,563,055-   U.S. Pat. No. 5,580,859-   U.S. Pat. No. 5,589,466-   U.S. Pat. No. 5,610,042-   U.S. Pat. No. 5,656,610-   U.S. Pat. No. 5,670,488-   U.S. Pat. No. 5,702,932-   U.S. Pat. No. 5,736,524-   U.S. Pat. No. 5,739,018-   U.S. Pat. No. 5,780,448-   U.S. Pat. No. 5,789,215,-   U.S. Pat. No. 5,824,544-   U.S. Pat. No. 5,830,725-   U.S. Pat. No. 5,849,304-   U.S. Pat. No. 5,851,826-   U.S. Pat. No. 5,858,744-   U.S. Pat. No. 5,871,982-   U.S. Pat. No. 5,871,983-   U.S. Pat. No. 5,871,986-   U.S. Pat. No. 5,879,934-   U.S. Pat. No. 5,888,502-   U.S. Pat. No. 5,925,565-   U.S. Pat. No. 5,928,906-   U.S. Pat. No. 5,932,210-   U.S. Pat. No. 5,935,819-   U.S. Pat. No. 5,945,100-   U.S. Pat. No. 5,955,331-   U.S. Pat. No. 5,981,274-   U.S. Pat. No. 5,994,136-   U.S. Pat. No. 5,994,624-   U.S. Pat. No. 6,013,516-   Ahmad et al., Dev. Biol., 194:86-98, 1998.-   Almendro et al., J. Immunol., 157(12):5411-5421, 1996.-   Alves da Costa et al., J. Neurochem., 90:800-806, 2004.-   Amado and Chen, Science, 285(5428):674-676, 1999.-   Angel et al., Cell, 49:729, 1987b.-   Angel et al., Mol. Cell. Biol., 7:2256, 1987a.-   Armentano et al., Proc. Natl. Acad. Sci. USA, 87(16):6141-6145,    1990.-   Artavanis-Tsakonas et al., Science, 284:770-776, 1999.-   Atchison and Perry, Cell, 46:253, 1986.-   Atchison and Perry, Cell, 48:121, 1987.-   Ausubel et al., In: Current Protocols in Molecular Biology, John,    Wiley & Sons, Inc, New York, 1994.-   Banerji et al., Cell, 27(2 Pt 1):299-308, 1981.-   Banerji et al., Cell, 33(3):729-740, 1983.-   Barany and Merrifield, In: The Peptides, Gross and Meienhofer    (Eds.), Academic Press, NY, 1-284, 1979.-   Barry et al., J. Biol. Chem., 276:15537-15546, 2001.-   Bates, Mol. Biotechnol., 2(2):135-145, 1994.-   Batra et al., Am. J. Respir. Cell Mol. Biol., 21(2):238-245, 1999.-   Battraw and Hall, Theor. App. Genet., 82(2):161-168, 1991.-   Beatus and Lendahl, J. Neurosci. Res., 54:125-136, 1998.-   Bellavia et al., Embo. J, 19:3337-3348, 2000.-   Bellavia et al., Proc. Natl. Acad. Sci. USA, 99:3788-3793, 2002.-   Berkhout et al., Cell, 59:273-282, 1989.-   Berset et al., Science, 291:1055-1058, 2001.-   Bett et al., J. Virololgy, 67(10):5911-5921, 1993.-   Bhattacharjee et al., J. Plant Bioch. Biotech., 6(2):69-73. 1997.-   Bilbao et al., Transplant Proc., 31(1-2):792-793, 1999.-   Blackwell et al., Arch. Otolaryngol. Head. Neck Surg.,    125(8):856-863, 1999.-   Blanar et al, EMBO J., 8:1139, 1989.-   Blomer et al., J. Virol., 71(9):6641-6649, 1997.-   Bodine and Ley, EMBO J., 6:2997, 1987.-   Boshart et al., Cell, 41:521, 1985.-   Bosze et al., EMBO J., 5(7):1615-1623, 1986.-   Braddock et al., Cell, 58:269, 1989.-   Brondello et al., J. Biol. Chem., 272:1368-1376, 1997.-   Bulla and Siddiqui, J. Virol., 62:1437, 1986.-   Callahan and Egan, J. Mammary Gland Biol. Neoplasia., 9:145-163,    2004.-   Campbell and Villarreal, Mol. Cell. Biol., 8:1993, 1988.-   Campbell et al., Am. Rev. Respir. Dis., 130(3):417-423, 1984.-   Campere and Tilghman, Genes and Dev., 3:537, 1989.-   Campo et al., Nature, 303:77, 1983.-   Campos et al., Circ. Res., 91:999-1006, 2002.-   Capaldi et al., Biochem. Biophys. Res. Comm., 76:425, 1977.-   Caplen et al., Gene Ther., 6(3):454-459, 1999.-   Carbonelli et al., FEMS Microbiol Lett., 177(1):75-82, 1999.-   Case et al., Proc. Natl. Acad. Sci. USA, 96(6):2988-2893, 1999.-   Celander and Haseltine, J. Virology, 61:269, 1987.-   Celander et al., J. Virology, 62:1314, 1988.-   Chandler et al., Cell, 33:489, 1983.-   Chandler et al., Proc. Natl. Acad. Sci. USA, 94(8):3596-601, 1997.-   Chang et al., Mol. Cell. Biol., 9:2153, 1989.-   Chatterjee et al., Proc. Natl. Acad. Sci. USA, 86:9114, 1989.-   Chen and Okayama, Mol. Cell. Biol. 7:2745-2752, 1987.-   Chen et al., Genes Dev., 10:2438-2451, 1996.-   Chillon et al., J. Virol., 73(3):2537-2540, 1999.-   Christou et al., Proc. Natl. Acad. Sci. USA, 84(12):3962-3966, 1987.-   Clay et al., Pathol. Oncol. Res., 5(1):3-15, 1999.-   Cocea, Biotechniques, 23(5):814-816, 1997.-   Coffey et al., Science, 282(5392):1332-1334, 1998.-   Cohen et al., J. Cell. Physiol., 5:75, 1987.-   Cook et al., Cell, 27:487-496, 1981.-   Costa et al., Mol. Cell. Biol., 8:81, 1988.-   Cripe et al., EMBO J., 6:3745, 1987.-   Culotta and Hamer, Mol. Cell. Biol., 9:1376, 1989.-   Culver et al., Science, 256(5063):1550-1552, 1992.-   Curry et al., Oncogene, 24:6333-6344, 2005.-   D'Halluin et al., Plant Cell, 4(12):1495-1505, 1992.-   Dandolo et al., J. Virology, 47:55-64, 1983.-   Dang et al., J. Natl. Cancer Inst., 92:1355-1357, 2000.-   De Villiers et al., Nature, 312(5991):242-246, 1984.-   DeLuca et al., J. Virol., 56(2):558-570, 1985.-   Deng et al., Cell, 82:675-684, 1995.-   Derby et al., Hear Res., 134(1-2):1-8, 1999.-   Deschamps et al., Science, 230:1174-1177, 1985.-   Domenga et al., Genes Dev., 18:2730-2735, 2004.-   Dorai et al., Int. J. Cancer, 82(6):846-852, 1999.-   Dovey et al., J. Neurochem., 76:173-181, 2001.-   Edbrooke et al., Mol. Cell. Biol., 9:1908, 1989.-   Ellisen et al., Cell, 66:649-661, 1991.-   Engel and Kohn, Front Biosci., 4:e26-33, 1999.-   EPO 0273085-   Faux et al., J. Neurosci., 21:5587-5596, 2001.-   Fechheimer, et al., Proc Natl. Acad. Sci. USA, 84:8463-8467, 1987.-   Feldman et al., Semin. Interv. Cardiol., 1(3):203-208, 1996.-   Feng and Holland, Nature, 334:6178, 1988.-   Feng et al., Nat. Biotechnol., 15(9):866-870, 1997.-   Firak and Subramanian, Mol. Cell. Biol., 6:3667, 1986.-   Fisher et al., Virology, 217(1):11-22, 1996.-   Fitzgerald et al., Oncogene, 19:4191-4198, 2000.-   Foder et al., Science, 251:767-773, 1991.-   Foecking and Hofstetter, Gene, 45(1):101-105, 1986.-   Forster and Symons, Cell, 49:211-220, 1987.-   Fraley et al., Proc. Natl. Acad. Sci. USA, 76:3348-3352, 1979.-   Freifelder, In: Physical Biochemistry Applications to Biochemistry    and Molecular Biology, 2nd Ed. Wm. Freeman and Co., NY, 1982.-   Frohman, In: PCR Protocols: A Guide To Methods And Applications,    Academic Press, N.Y., 1990.-   Fujita et al., Cell, 49:357, 1987.-   Fujiwara and Tanaka, Nippon Geka Gakkai Zasshi, 99(7):463-468, 1998.-   Garoff and Li, Curr. Opin. Biotechnol., 9(5):464-469, 1998.-   Gamido et al., J. Neurovirol., 5(3):280-288, 1999.-   Gefter et al., Somatic Cell Genet., 3:231-236, 1977.-   Gerlach et al., Nature (London), 328:802-805, 1987.-   Ghosh and Bachhawat, In: Liver Diseases, Targeted Diagnosis and    Therapy Using Specific Receptors and Ligands, Wu and Wu (Eds.),    Marcel Dekker, New York, 87-104, 1991.-   Gilles et al., Cell, 33:717, 1983.-   Glasser et al., Am. J. Physiol., 261:L349-356, 1991.-   Gloss et al., EMBO J., 6:3735, 1987.-   Gnant et al., Cancer Res., 59(14):3396-403, 1999.-   Gnant et al., J. Natl. Cancer Inst., 91(20):1744-1750, 1999.-   Godbout et al., Mol. Cell. Biol., 8:1169, 1988.-   Goding, In: Monoclonal Antibodies: Principles and Practice, 2d ed.,    Orlando, Fla., Academic Press, 60-61, 65-66, 71-74, 1986.-   Goodbourn and Maniatis, Proc. Natl. Acad. Sci. USA, 85:1447, 1988.-   Goodbourn et al., Cell, 45:601, 1986.-   Gopal, Mol. Cell. Biol., 5:1188-1190, 1985.-   Graham and Prevec Mol. Biotechnol., 3(3):207-220, 1995.-   Graham and Van Der Eb, Virology 52:456-467, 1973-   Greene et al., Immunology Today, 10:272, 1989-   Grosschedl and Baltimore, Cell, 41:885, 1985.-   Hacia et al., Nature Genet., 14:441-449, 1996.-   Haecker et al., Hum. Gene Ther., 7(15):1907-1914, 1996.-   Han et al., Euro. J Surgical Oncology, 25:194-198, 1999.-   Haneda et al., Eur. J. Pharmacol., 365:1-7, 1999.-   Harland and Weintraub, J. Cell Biol., 101: 1094-1099, 1985.-   Harlow and Lane, In: Antibodies: A laboratory Manual, Cold Spring    Harbor Laboratories, Cold Spring Harbor, N.Y., 1988.-   Haruki et al., Cancer Res., 65:3555-3561, 2005.-   Haslinger and Karin, Proc. Natl. Acad. Sci. USA, 82:8572, 1985.-   Hauber and Cullen, J. Virology, 62:673, 1988.-   Hayashi et al., Neurosci. Lett., 267(1):37-40, 1999.-   He et al., Plant Cell Reports, 14 (2-3):192-196, 1994.-   Hen et al., Nature, 321:249, 1986.-   Hensel et al., Lymphokine Res., 8:347, 1989.-   Hermens and Verhaagen, Prog. Neurobiol., 55(4):399-432, 1998.-   Herr and Clarke, Cell, 45:461, 1986.-   Hirochika et al., J. Virol., 61:2599, 1987.-   Hirsch et al., Br. J. Cancer, 86:1449-1456, 2002.-   Hirsch et al., Mol. Cell. Biol., 10:1959, 1990.-   Hogan et al., In: Manipulating the Mouse Embryo: A Laboratory    Manual, 2nd ed., Cold Spring Harbor Laboratory Press, 1994.-   Holbrook et al., Virology, 157:211, 1987.-   Holley, Nature, 258:487-490, 1975.-   Holzer et al., Virology, 253(1):107-114, 1999.-   Horlick and Benfield, Mol. Cell. Biol., 9:2396, 1989.-   Hou and Lin, Plant Physiology, 111:166, 1996.-   Howard et al., Ann. NY Acad. Sci., 880:352-365, 1999.-   Hrabe de Angelis et al., Nature, 386:717-721, 1997.-   Huang et al., Cell, 27:245, 1981.-   Huard et al., Neuromuscul Disord., 7(5):299-313, 1997.-   Hug et al., Mol. Cell. Biol., 8:3065, 1988.-   Hwang et al., Mol. Cell. Biol., 10:585, 1990.-   Imagawa et al., Cell, 51:251, 1987.-   Imai et al., J. Virol., 72(5):4371-4378, 1998.-   Imbra and Karin, Nature, 323:555, 1986.-   Imler et al., Mol. Cell. Biol., 7:2558, 1987.-   Imperiale and Nevins, Mol. Cell. Biol., 4:875, 1984.-   Innis et al., Proc Natl Acad Sci USA, 85(24):9436-9440, 1988.-   Irie et al., Antisense Nucleic Acid Drug Dev., 9(4):341-349, 1999.-   Jakobovits et al., Mol. Cell. Biol., 8:2555, 1988.-   Jameel and Siddiqui, Mol. Cell. Biol., 6:710, 1986.-   Jaynes et al., Mol. Cell. Biol., 8:62, 1988.-   Jemal et al., CA Cancer J. Clin., 55:10-30, 2005.-   Jhappan et al., Genes Dev., 6:345-355, 1992.-   Johnson et al., IN: Biotechnology And Pharmacy, Pezzuto et al.,    (Eds.), Chapman and Hall, New York, 1993.-   Johnson et al., Mol. Cell. Biol., 9:3393, 1989.-   Johnston and Edgar, Nature, 394:82-84, 1998.-   Johnston et al., J. Virol., 73(6):4991-5000, 1999.-   Joutel et al., J. Clin. Invest., 105:597-605, 2000.-   Joyce, Nature, 338:217-244, 1989.-   Kadesch and Berg, Mol. Cell. Biol., 6:2593, 1986.-   Kaeppler et al., Plant Cell Reports, 9:415-418, 1990.-   Kaneda et al., Science, 243:375-378, 1989.-   Karin et al., Mol. Cell. Biol., 7:606, 1987.-   Katinka et al., Cell, 20:393, 1980.-   Kato et al, J. Biol. Chem., 266(6):3361-3364, 1991.-   Kaufman et al., Surv. Opthalmol., 43 Suppl 1: S91-97, 1999.-   Kawamoto et al., Mol. Cell. Biol., 8:267, 1988.-   Kay, Haemophilia, 4(4):389-392, 1998.-   Kiledjian et al., Mol. Cell. Biol., 8:145, 1988.-   Kim and Cech, Proc. Natl. Acad. Sci. USA, 84:8788-8792, 1987.-   Klamut et al., Mol. Cell. Biol., 10: 193, 1990.-   Klimatcheva et al., Front Biosci., 4:D481-96, 1999.-   Koch et al., Mol. Cell. Biol., 9:303, 1989.-   Kohler and Milstein, Eur. J. Immunol., 6:511-519, 1976.-   Kohler and Milstein, Nature, 256:495-497, 1975.-   Kohut et al., Am. J. Physiol., 275(6 Pt 1):L1089-94, 1998.-   Kooby et al., FASEB J, 13(11):1325-1334, 1999.-   Kornberg, In: DNA Replication, W. H. Freeman and Company, New York,    1992.-   Kraus et al., FEBS Lett., 428(3):165-170, 1998.-   Krebs et al., Genes Dev., 14:1343-1352, 2000.-   Kriegler and Botchan, In: Eukaryotic Viral Vectors, Gluzman (Ed.),    Cold Spring Harbor: Cold Spring Harbor Laboratory, NY, 1982.-   Kriegler and Botchan, Mol. Cell. Biol., 3:325, 1983.-   Kriegler et al., Cell, 38:483, 1984.-   Kriegler et al., Cell, 53:45, 1988.-   Krisky et al., Gene Ther, 5(11):1517-1530, 1998a.-   Krisky et al., Gene Ther., 5(12):1593-1603, 1998b.-   Kuhl et al., Cell, 50:1057, 1987.-   Kunz et al., Nucl. Acids Res., 17:1121, 1989.-   Kwoh et al., Proc. Natl. Acad. Sci. USA, 86(4):1173-1177, 1989.-   Kyte and Doolittle, J. Mol. Biol., 157(1):105-132, 1982.-   Lachmann and Efstathiou, Clin. Sci. (Colch), 96(6):533-541, 1999.-   Lanford et al., Nat. Genet., 21:289-292, 1999.-   Lardelli et al., Mech Dev., 59:177-190, 1996.-   Lareyre et al., J. Biol. Chem., 274(12):8282-8290, 1999.-   Larsen et al., Proc Natl. Acad. Sci. USA., 83:8283, 1986.-   Laspia et al., Cell, 59:283, 1989.-   Latimer et al., Mol. Cell. Biol., 10:760, 1990.-   Lazzeri, Methods Mol. Biol., 49:95-106, 1995.-   Lee et al., J. Auton. Nerv. Syst., 74(2-3):86-90, 1997.-   Lee et al., Korean J. Genet., 11(2):65-72, 1989.-   Lee et al., Nature, 294:228, 1981.-   Lee et al., Nature, 329(6140):642-645, 1987.-   Lee et al., Nucleic Acids Res., 12:4191-206, 1984.-   Leibowitz et al., Diabetes, 48(4):745-753, 1999.-   Leonhardt et al., J. Cell Biol., 149:271-280, 2000.-   Lesch, Biol. Psychiatry, 45(3):247-253, 1999.-   Levenson et al., Human Gene Therapy, 9:1233-1236, 1998.-   Levinson et al., Nature, 295:79, 1982.-   Levitan and Greenwald, Nature, 377:351-354, 1995.-   Li et al., Science, 275:1943-1947, 1997.-   Liang and Pardee, Nature Reviews Cancer, 3:869-876, 2003.-   Liang, Biotechniques, 33:338-346, 2002.-   Lin et al., Mol. Cell. Biol., 10:850, 1990.-   Linggi et al., Oncogene, 25:160-163, 2006.-   Lu et al., Clin. Cancer Res., 10:3291-3300, 2004.-   Lundstrom, J. Recept. Signal Transduct. Res., 19(1-4):673-686, 1999.-   Luria et al., EMBO J., 6:3307, 1987.-   Lusky and Botchan, Proc. Natl. Acad. Sci. USA, 83:3609, 1986.-   Lusky et al., Mol. Cell. Biol., 3:1108, 1983.-   Ma et al., Hematol. Oncol., 17:91-105, 1997.-   Macejak and Sarnow, Nature, 353:90-94, 1991.-   Magi-Galluzzi et al., Lab Invest., 76:37-51, 1997.-   Majors and Varmus, Proc. Natl. Acad. Sci. USA, 80:5866, 1983.-   Marienfeld et al., Gene Ther., 6(6): 1101-1113, 1999.-   Mastrangelo et al., Cancer Gene Ther., 6(5):409-422 1999.-   McNeall et al., Gene, 76:81, 1989.-   Meert et al., Eur. Respir. J., 20:975-981, 2002.-   Merrifield, Science, 232(4748):341-347 1986.-   Michel and Westhof, J. Mol. Biol., 216:585-610, 1990.-   Miksicek et al., Cell, 46:203, 1986.-   Miller et al., Methods Enzymol., 217:581-599, 1993.-   Mitsiadis et al. Dev. Biol., 204:420-431, 1998.-   Miyamoto et al., Cancer Cell, 3:565-576, 2003.-   Miyatake et al., Gene Ther., 6(4):564-572, 1999.-   Moldawer et al., Shock, 12(2):83-101, 1999.-   Mordacq and Linzer, Genes and Dev., 3:760, 1989.-   Moreau et al., Nucl. Acids Res., 9:6047, 1981.-   Moriuchi et al., Cancer Res., 58(24):5731-5737, 1998.-   Morrison et al., J. Gen. Virol., 78(Pt 4):873-878, 1997.-   Muesing et al., Cell, 48:691, 1987.-   Mumm and Kopan, Dev. Biol., 228:151-165, 2000.-   Nahle et al., Nat. Cell Biol., 4:859-864, 2002.-   Naldini et al., Proc. Natl. Acad. Sci. USA, 93(21):11382-11388,    1996.-   Neumann et al., Proc. Natl. Acad. Sci. USA, 96(16):9345-9350, 1999.-   Ng et al., Nuc. Acids Res., 17:601, 1989.-   Nicolau and Sene, Biochim. Biophys. Acta, 721:185-190, 1982.-   Nicolau et al., Methods Enzymol., 149:157-176, 1987-   Nomoto et al., Gene, 236(2):259-271, 1999.-   Ohara et al., Proc. Natl. Acad. Sci. USA, 86:5673-5677, 1989.-   Omirulleh et al., Plant Mol. Biol., 21(3):415-28, 1993.-   Ondek et al., EMBO J., 6:1017, 1987.-   Ornitz et al., Mol. Cell. Biol., 7:3466, 1987.-   Oswald et al., Mol. Cell. Biol., 18:2077-2088, 1998.-   Palmiter et al., Nature, 300:611, 1982.-   Paris et al., Eur. J. Pharmacol., 514:1-15, 2005.-   Parks et al., J. Virol., 71(4):3293-8, 1997.-   PCT Appln. WO 9217598-   PCT Appln. WO 94/09699-   PCT Appln. WO 95/06128-   Pear et al., J. Exp. Med., 183:2283-2291, 1996.-   Pease et al., Proc. Natl. Acad. Sci. USA, 91:5022-5026, 1994.-   Pech et al., Mol. Cell. Biol., 9:396, 1989.-   Pelletier and Sonenberg, Nature, 334:320-325, 1988.-   Pelletier et al., Cancer Res., 66:3681-3687, 2006.-   Perales et al., Proc. Natl. Acad. Sci. USA, 91:4086-4090, 1994.-   Perez-Stable and Constantini, Mol. Cell. Biol., 10:1116, 1990.-   Petrof, Eur. Respir. J., 11(2):492-497, 1998.-   Picard and Schaffner, Nature, 307:83, 1984.-   Pignon J et al., Hum. Mutat., 3(2):126-132, 1994.-   Pinkert et al., Genes and Dev., 1:268, 1987.-   Plows et al., Biochem J., 362:305-315, 2002.-   Polyak et al., Genes Dev., 10:1945-1952, 1996.-   Ponta et al., Proc. Natl. Acad. Sci. USA, 82:1020, 1985.-   Porton et al., Mol. Cell. Biol., 10: 1076, 1990.-   Potrykus et al., Mol. Gen. Genet., 199:183-188, 1985.-   Potter et al., Proc. Natl. Acad. Sci. USA, 81:7161-7165, 1984.-   Purow et al., Cancer Res., 65:2353-2363, 2005.-   Qin et al., Mol. Cancer. Ther., 3:895-902, 2004.-   Queen and Baltimore, Cell, 35:741, 1983.-   Quinn et al., Mol. Cell. Biol., 9:4713, 1989.-   Rabinovitch et al., Diabetes, 48(6):1223-1229, 1999.-   Rebay et al., Cell, 74:319-329, 1993.-   Reddy et al., J. Virol., 72(2):1394-1402, 1998.-   Redondo et al., Science, 247:1225, 1990.-   Reinhold-Hurek and Shub, Nature, 357:173-176, 1992.-   Reisman and Rotter, Mol. Cell. Biol., 9:3571, 1989.-   Remington's Pharmaceutical Sciences, 15^(th) Ed., 33:624-652, 1990.-   Remington's Pharmaceutical Sciences, 15^(th) Ed., 1035-1038 and    1570-1580, 1990.-   Resendez Jr. et al., Mol. Cell. Biol., 8:4579, 1988.-   Rhodes et al., Methods Mol. Biol., 55:121-131, 1995.-   Ripe et al., Mol. Cell. Biol., 9:2224, 1989.-   Rippe et al., Mol. Cell. Biol., 10:689-695, 1990.-   Rittling et al., Nuc. Acids Res., 17:1619, 1989.-   Robbins and Ghivizzani, Pharmacol. Ther., 80(1):35-47, 1998.-   Robbins et al., Proc. Natl. Acad. Sci. USA, 95(17):10182-10187 1998.-   Robbins et al., Trends Biotechnol., 16(1):35-40, 1998.-   Robey et al., Cell, 87:483-492, 1996.-   Rodenhuis et al., J. Clin. Oncol., 15:285-291, 1997.-   Rohn et al., J. Virol., 70:8071-8080, 1996.-   Rosen et al., Cell, 41:813, 1988.-   Sakai et al., Genes and Dev., 2:1144, 1988.-   Sambrook et al., In: Molecular Cloning: A Laboratory Manual, Cold    Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,    1(7):7.19-17.29, 1989.-   Santagata et al., Cancer Res., 64:6854-6857, 2004.-   Sarver et al., Science, 247:1222-1225, 1990.-   Satake et al., J. Virology, 62:970, 1988.-   Sawai et al., Mol. Genet. Metab., 67(1):36-42, 1999.-   Scanlon et al., Proc. Natl. Acad. Sci. USA, 88:10591-10595, 1991.-   Schaffner et al., J. Mol. Biol., 201:81, 1988.-   Searle et al., Mol. Cell. Biol., 5:1480, 1985.-   Sharp and Marciniak, Cell, 59:229, 1989.-   Shaul and Ben-Levy, EMBO J., 6:1913, 1987.-   Shelly et al., J. Cell Biochem., 73:164-175, 1999.-   Sherman et al., Mol. Cell. Biol., 9:50, 1989.-   Shimizu et al., Mol. Cell. Biol., 20:6913-6922, 2000.-   Shoemaker et al., Nature Genetics, 14:450-456, 1996.-   Sivaraman et al., J. Clin. Invest., 99:1478-1483, 1997.-   Sleigh and Lockett, J. EMBO, 4:3831, 1985.-   Small et al., J. Biol. Chem., 278:16405-16413, 2003.-   Smith, Arch. Neurol., 55(8):1061-1064, 1998.-   Spalholz et al., Cell, 42:183, 1985.-   Spandau and Lee, J. Virology, 62:427, 1988.-   Spandidos and Wilkie, EMBO J., 2:1193, 1983.-   Stambolic et al., Mol. Cell, 8:317-325, 2001.-   Steck et al., Nat. Genet., 15:356-362, 1997.-   Stein et al., J. Biol. Chem., 279:48930-48940, 2004.-   Stephens and Hentschel, Biochem. J, 248:1, 1987.-   Stewart and Young, In: Solid Phase Peptide Synthesis, 2^(nd) Ed.,    Pierce Chemical Co., 1984.-   Stewart et al., Gene Ther., 6(3):350-363, 1999.-   Stuart et al., Nature, 317:828, 1985.-   Sullivan and Peterlin, Mol. Cell. Biol., 7:3315, 1987.-   Sundaram, Genes Dev., 19:1825-1839, 2005.-   Suzuki et al., Biochem. Biophys. Res. Commun., 252(3):686-690, 1998.-   Swartzendruber and Lehman, J. Cell. Physiology, 85:179, 1975.-   Sweeney et al., Faseb. J., 18:1421-1423, 2004.-   Taichman et al., Dev. Dyn., 225:166-175, 2002.-   Takebe et al., Mol. Cell. Biol., 8:466, 1988.-   Tam et al., J. Am. Chem. Soc., 105:6442, 1983.-   Tanaka et al., Oncogene, 8:2253-2258, 1993.-   Taniura et al., J. Biol. Chem., 274:16242-16248, 1999.-   Tavernier et al., Nature, 301:634, 1983.-   Taylor and Kingston, Mol. Cell. Biol., 10:165, 1990a.-   Taylor and Kingston, Mol. Cell. Biol, 10:176, 1990b.-   Taylor and Stark, Oncogene, 20:1803-1815, 2001.-   Taylor et al., J. Biol. Chem., 264:15160, 1989.-   Thiesen et al., J. Virology, 62:614, 1988.-   Timiryasova et al., Int. J. Oncol., 14(5):845-854, 1999.-   Timiryasova et al., Oncol. Res.; 11(3):133-144, 1999.-   Traverse et al., Biochem. J, 288 (Pt 2):351-355, 1992.-   Treisman, Cell, 42:889, 1985.-   Tronche et al., Mol. Biol. Med., 7:173, 1990.-   Trudel and Constantini, Genes and Dev., 6:954, 1987.-   Tsukada et al., Plant Cell Physiol., 30(4)599-604, 1989.-   Tsumaki et al., J. Biol. Chem., 273(36):22861-22864, 1998.-   Tur-Kaspa et al., Mol. Cell. Biol., 6:716-718, 1986.-   Tyndell et al., Nuc. Acids. Res., 9:6231, 1981.-   Vanderkwaak et al., Gynecol. Oncol., 74(2):227-234, 1999.-   Vannice and Levinson, J. Virology, 62:1305, 1988.-   Vasseur et al., Proc Natl. Acad. Sci. USA, 77:1068, 1980.-   Vogelstein et al., Nature, 408(6810):307-310, 2000.-   Vogelstein, Nature, 348(6303):681-682, 1990.-   Vousden and Lu, Nat. Rev. Cancer, 2:594-604, 2002.-   Vousden and Prives, Cell, 120:7-10, 2005.-   Wagner et al., Science, 260:1510-1513, 1990.-   Walker et al., Nucleic Acids Res., 20(7):1691-1696, 1992.-   Wang and Calame, Cell, 47:241, 1986.-   Wang et al., Gynecol. Oncol., 71(2):278-287, 1998.-   Wang et al., J. Biol. Chem., 277:23165-23171, 2002.-   Weber et al., Cell, 36:983, 1984.-   Weihl et al., Neurosurgery, 44(2):239-252, 1999.-   Weinberg et al., Biochemistry, 28:8263-8269, 1989.-   Weinberger et al., Mol. Cell. Biol., 8:988, 1984.-   Weng et al., Science, 306:269-271, 2004.-   White et al., J. Virol., 73(4):2832-28340, 1999.-   Williams et al., Blood, 107(3):931-939, 2006.-   Wilson, J. Clin. Invest., 98(11):2435, 1996.-   Winoto and Baltimore, Cell, 59:649, 1989.-   Wolfe and Kopan, Science, 305:1119-1123, 2004.-   Wong et al., Gene, 10:87-94, 1980.-   Wu and Wallace, Genomics, 4:560-569, 1989.-   Wu and Wu, Adv. Drug Delivery Rev., 12:159-167, 1993.-   Wu and Wu, J. Biol. Chem., 262:4429-4432, 1987.-   Wu et al., Biochem. Biophys. Res. Commun., 233(1):221-226, 1997.-   Wu, Chung Hua Min Kuo Hsiao Erh Ko I Hsueh Hui Tsa Chih,    39(5):297-300, 1998.-   Xue et al., Hum Mol Genet., 8:723-730, 1999.-   Yamada et al., Proc. Natl. Acad. Sci. USA, 96(7):4078-4083, 1999.-   Yang and Liang, Mol. Biotechnol., 3:197-208, 2004.-   Yeung et al., Gene Ther., 6(9):1536-1544, 1999.-   Yoo et al., Science, 303:663-666, 2004.-   Yoon et al., J. Gastrointest. Surg., 3(1):34-48, 1999.-   Yu and Zhang, Biochem. Biophys. Res. Commun., 331:851-858, 2005.-   Yu et al., Proc. Natl. Acad. Sci. USA, 100:1931-1936, 2003.-   Yu et al., Proc. Natl. Acad. Sci. USA, 96:14517-14522, 1999.-   Yutzey et al., Mol. Cell. Biol., 9:1397, 1989.-   Zeng et al., Cancer Cell, 8:13-23, 2005.-   Zhao-Emonet et al., Biochim. Biophys. Acta, 1442(2-3):109-119, 1998.-   Zheng et al., J. Gen. Virol., 80(Pt 7):1735-1742, 1999.-   Zhou et al., Exp. Hematol, 21:928-933, 1993.-   Zufferey et al., Nat. Biotechnol., 15(9):871-875, 1997.

1-31. (canceled)
 32. An isolated and purified antibody that binds to anepitope comprising a sequence selected from the group consisting ofCFNTLGGHS (SEQ ID NO:3), CVCVNGWTGES (SEQ ID NO:4), CATAV (SEQ ID NO:5),CFHGAT (SEQ ID NO:6), CVSNP (SEQ ID NO:7) and CLNGGS (SEQ ID NO:8). 33.A method of inhibiting Notch3 receptor signaling comprising contacting acell expressing Notch3 with an antibody that binds to an epitopecomprising a sequence selected from the group consisting of CFNTLGGHS(SEQ ID NO:3), CVCVNGWTGES (SEQ ID NO:4), CATAV (SEQ ID NO:5), CFHGAT(SEQ ID NO:6), CVSNP (SEQ ID NO:7) and CLNGGS (SEQ ID NO:8).
 34. Amethod of treating a subject having a Notch3-expressing cancercomprising administering to said subject an antibody that binds to anepitope comprising a sequence selected from the group consisting ofCFNTLGGHS (SEQ ID NO:3), CVCVNGWTGES (SEQ ID NO:4), CATAV (SEQ ID NO:5),CFHGAT (SEQ ID NO:6), CVSNP (SEQ ID NO:7) and CLNGGS (SEQ ID NO:8). 35.A pharmaceutical formulation comprising an antibody that binds to anepitope comprising a sequence selected from the group consisting ofCFNTLGGHS (SEQ ID NO:3), CVCVNGWTGES (SEQ ID NO:4), CATAV (SEQ ID NO:5),CFHGAT (SEQ ID NO:6), CVSNP (SEQ ID NO:7) and CLNGGS (SEQ ID NO:8). 36.(canceled)